مغز فراری

دست نامریی

2008-05-22T12:12:00.004-04:00

یادش بخیر دوستی داشتم که الان حدود 3 سال است که به کانادا مهاجرت کرده و حدود 5 سال باهم همکار و هم اتاقی بودیم. ایشان ... بودند و به خیلی از چیزهایی که ما معتقدیم ، اعتقادی نداشتند ولی یک مطلب جالبی در مورد وجود امام زمان می گفتند که خیلی جالب بود.

ایشان همیشه به من می گفتند که تو خودت میدونی که من به خیلی چیزها اعتقادی ندارم ولی به وجود امام زمان اعتقاد دارم چون کشوری که بدون هیچ حساب و کتابی و با اینهمه مسوول بی لیاقت با بدترین نوع مدیریت هنوز هم به حیات خودش ادامه می دهد معلوم است که یک دست نامریی آنرا اداره می کند و الا هیچ کشوری با این وضعیت قادر به ادامه حیات نمی باشد. ایشون می گفتند که به نظر من آن دست نامریی متعلق به امام زمان می باشد که این مملکت را اداره می کند.

Ref:http://ausimigrant.blogspot.com/2008/02/blog-post_8271.html

فرار مغزها - مهاجرت به استرالیا

زندگی در کدام سو

2008-05-22T11:59:00.004-04:00

نیک‌آهنگ کوثر:

چند روز پیش با دوستانم در تهران گپ می‌زدم. بحث زندگی در ایران بود. یکهو به فکرم رسید که بپرسم واقعا کجا دوست دارید زندگی کنید؟ راستش را بخواهید، من تقریبا هر شب خواب تهران را می‌بینم. دلم برای بوی گازوئیل هم تنگ شده! اما آیا واقعا دوست دارم در تهران زندگی کنم با تمامی محدودیت‌ها؟ آری و نه! ایران مثل خانه پدری می‌ماند. دوستش داشته‌ای، کلی خاطره... ولی حاضر نیستی به آن برای زندگی برگردی! می‌خواهی چه کاری داشته باشی؟ چه امنیتی؟ چه تامینی؟ اصلا بحث خودباختگی نیست! من الان بعد آز ۸ ساعت کار سخت کانادایی، برگشته‌ام خانه، لپ‌تاپم روی پایم است - مرتبط با اینترنت مسدود نشده - و دارم بدون نگرانی، اخبار آمریکایی نگاه می‌کنم و حوصله‌ام که سر رفت، می‌روم ۷ کانال فیلم قانونی را نگاه می‌کنم و ... اینجا وطن من نبوده، ولی احساس می‌کنم قفسی است که خودم انتخابش کرده‌ام. صدایم را در فضای بسته‌ام کوتاه نمی‌کنم تا شنود کننده‌ها نشوند و بعدا برایم نوارش را بگذارند. قیمت گوشت و برنج و خیلی چیزها اینجا ارزان‌تر است! مالیات وحشتناکی می‌دهیم، ولی اثراتش را هم می‌بینیم. از من که نه، از آرش کمانگیر در باره سرما بپرسید! برای خیلی‌ها غیر قابل تحمل بوده، اما آیا می‌شود سپری‌اش کرد؟ اینجا رقابت برسر کار وحشتناک است. یک خطا یا کوتاهی یا بدشانسی کافی است که شغلت بپرد. بدشانسی چرا؟ بازار بورس! افت ارزش پول! بحران‌های اعتباری و ... ولی وقتی شرایط کاری ایران و تعطیلی راحت رسانه های غیر دولتی را برای جماعت کانادایی تعریف می‌کنی، شاخ در می‌آورند! سوال من این است: آیا واقعا در صورت فراهم بودن شرایط برای مهاجرت، حاضر به تحمل وضع موجود در داخل کشور هستید یا نه؟
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من یکی که 7 سالی میشه از تهرون هم پامو بیرون نذاشتم! حالا نمیدونم چرا به این لینک مثبت دادم چون ربطی به امثال من که نداره!:) حالا اینجا همچین سخت هم نیست ها...سختیش همون 50 سال اوله بعد خوب میشه!

افرادی که از داخل ایران به این سوال جواب می دن خیلی عمق سوال را نمی فهمند. ما هم که اینقدر در عمقش فرو رفتیم که غرق شدیم و توانایی جواب دادن نداریم.

افرادی که از داخل ایران به این سوال جواب می دن خیلی عمق سوال را نمی فهمندکاملا" درسته...یه مجموعه ای قبلا" از سیما پخش میشد بنام مهاجران که توش زندگی ایرانیان موفق خارج از کشور نشون داده میشد.با یکی از اینها که صحبت میکرد به مغزش اشاره کرد و گفت: مهاجرت اینجا اتفاق میفته!...حالا احتمالا" جواب این سوال بستگی داره به اینکه مهاجرت تو مغز طرف اتفاق افتاده باشه یا صرفا" یه تغییر مکان فیزیکی بوده باشه!البته همونطور که فرمودید برای من که تو ایرانم درکش واقعا" دشواره.

من چهار سال است که مهاجرت کرده ام ... زندگی راحت و خیلی چیزهای دیگر را فدای آمدنم کردم ... در ایران روزی چهار ساعت کار می کردم ... کار هم که نمی شد گفت... برای من تفریح بود ... هفته ای چهار ساعت در دانشگاه تدریس می کردم و بقیه روزها را در یک مدرسه کار می کردم... البته درآمد چندانی هم نداشتم ولی می گذشت... اکنون در آمریکا یک کارگر هستم ... البته لقب manager را یدک می کشم ولی برای بقا باید بیش از کارگران عرق بریزم و کار بدنی بکنم. گاهی روزی بیش از 18 ساعت کار می کنم... آن هم کار با معیار های آمریکایی... سه روز گذشته را با درد ناشی از سنگ مثانه سر کار رفته ام و وانمود کرده ام ok هستم ...آیا راضی هستم؟ آری و نه... بیشتر از این توقع داشتم ولی نتوانستم ... شاید اگر مجرد بودم زندگی به گونه ای دیگر بود ولی ... آیا اگر دوباره در شرایط یکسان قرار بگیرم به آمریکا مهاجرت خواهم کرد؟ آری... بی تردبد... در اینجا از نفس کشیدن نمی ترسم... نفس کشیدن و زنده ماندن سخت هست ، اما ترس آور نیست... چهار سال است که به خودکشی نمی اندیشم ... دروغ چرا ... دیگر از آن می ترسم.

دلم برای ایران تنگ شده است... به ویژه تهران و کرج... گاهی در google earth جاهایی را که زمانی دوست داشتم مرور می کنم ... ساعت ها و ساعت ها نام دوستانم را در اینترنت جستجو می کنم تا شاید نشانی از ایشان بیابم ... اما دوست ندارم بازگردم... دست کم در این زمانه و تا این رژیم بر قرار است... مسخره است ... اما دوست ندارم برادرم بیاید ... دوست ندارم دوستانم بیایند... اگر می توانستم زندگی خوبی برای همه شان فراهم کنم آرزویم بود که بیایند... ولی اکنون نه... نمی دانم می توانند تاب بیاورند؟ آیا سختی ها خردشان نخواهد کرد؟ آرزوی قلبی ام این است که بیایند ولی مغزم ... ولی مغزم می گوید بگذار خودشان تصمیم بگیرند... سخت است ... خیلی سخت است ولی شاید ارزشش را داشته باشد.

ایران مثل خانه پدری می‌ماند. دوستش داشته‌ای، کلی خاطره... ولی حاضر نیستی به آن برای زندگی برگردی! So true :-)

کاربر nikagang شما دنبال جواب این سؤال، برای خود تان میگردید، یا دیگران.اگر برای دیگران است، که وجود 4 میلیونی ایرانی در خارج از کشور و سیلی که هنوز هم ادامه دارد، باید جوابی باندازه کافی قانع کننده باشه.اگرهم برای خود شماست، پس بنظر میرسد، که گم شدی، و همه کارهای نوشتنی و کشیدنی شما در عین زیبا و پر معنی بودن، برای مخاطبان شما، از نظر خود شما، بی هدف است.

من برای ادامه تحصیل از ایران زدم بیرون... آخرین ترم فوق لیسانسم رو دارم میگذرونم (MBA)، متاهل هم هستم.... هنوز 2 سال نشده که خارج از ایران زندگی میکنم، اما دارم لحظه شماری می کنم برگردم؛ خیلی ها اینو که میشنون خندشون میگیره... اما هیچکجای دنیا (حتی جایی که بهترین شرایط زندگی در اختیارت باشه) ایران نمیشه!مشکلات و گرفتاری و تورم و اعطاب خوردی و سیاست بازیهای مسخره و بگیروببندها و ...و ... همه رو چشیدیم، اما خیلی چیزهای دیگرو نمیشه بیخیالش شد. اونایی که این حس رو دارن نمیتونن دل بکنن.

افرادی که از داخل ایران به این سوال جواب می دن خیلی عمق سوال را نمی فهمند. ما هم که اینقدر در عمقش فرو رفتیم که غرق شدیم و توانایی جواب دادن نداریم.راست میگه مثلا همین iraneraha نفهمیده که چی شده تا وقتی مهاجرت نکنی نمیفهمی چی میگن مهاجرا با اینکه خیلی رک و واضح میگن چه کنیم ما هم اسیر روزمرگی هستیم و دل خوش به لب تاب
دلم برای ایران تنگ شده است... به ویژه تهران و کرج... گاهی در google earth جاهایی را که زمانی دوست داشتم مرور می کنم خدمت همشهری سابق خودم عرض کنم این google earth رو خوب اومدیدر کل ولی شاید من برگردم! با همه مشکلاتی مه ایران داره اگر توی ایران هم مثل اینجا کار کنم حتما به جایی میرسم اینجا یاد گرفتم که به فکر بیزینس خودم باشم و بر نامه آخر هفته همین زندگی مگه چیه اینو به یکی گفتم گفت ولی اگر برگردی نمیذارن که به فکر خودت باشی اینقدر چوب تو آستینت میکنند که دوباره مخت معیوب میشه و همه چی رو در فرار میبینی

این خانه قشنگ است ولی خانه من نیست این خاک چه زیباست ولی خاک وطن نیستآن دختـــــــــــر چشم آبی گیسوی طلایی طناز سیه چشــــــــــم چو معشوقه من نیستآن کشور نو آن وطــــن دانش و صنعت هرگز به دل انگیــــــــــزی ایران کهن نیستدر مشهد و یزد و قم و سمنان و لرستان لطفی است که در کلگری و نیس و پکن نیستدر دامن بحر خزر و ساحل گیلان موجی است که در ساحل دریای عدن نیستدر پیکر گلهای دلاویز شمیران عطری است که در نافه ی آهوی ختن نیستآواره ام و خسته و سرگشته و حیران هرجا که روم هیچ کجا خانه من نیستآوارگی وخانه به دوشی چه بلایست دردی است که همتاش در این دیر کهن نیستمن بهر که خوانم غزل سعدی و حافظ در شهر غریبی که در او فهم سخن نیستهرکس که زند طعنه به ایرانی و ایران بی شبه که مغزش به سر و روح به تن نیستپاریس قشنگ است ولی نیست چوتهران لندن به دلاویزی شیراز کهن نیست هر چند که سرسبز بود دامنه آلپ چون دامن البرز پر از چین وشکن نیستاین کوهبلند است ولی نیست دماوند این رود چه زیباست ولی رود تجن نیستاین شهرعظیم است ولی شهرغریب است این خانه قشنگ است ولی خانه من نیستدکتر خسرو فرشید ورد

پاریس قشنگ است ولی نیست چوتهرانلندن به دلاویزی شیراز کهن نیست

من که اگه بدونم در ایران برام کار هست حتما حتما بر میگردم !!! البته هنوز سال اول دانشکدم!!! یه ۴ سالی هست اومدیم !!! موقعی هم که فهمیدم داریم مهاجرت میکنیم اصلا هیجان زده نشدم!!!خداییش اینجا برام خسته کنندست!!! نه دوست انچنانی و نه هیج فک و فامیلی !!! هواش مثل زهر مار و با غذا هاش هم اصلا حال نمیکنم!!!درست ارامش داریم!!! هر روز نگران گرانی غذا نیستیم و خدا رو شکر یه سقفی بالای سر داریم!!!شاید روزی که برگردم برای خیلی چیزهای اینجا دلم تنگ بشه ولی خب ...

ساعت ها و ساعت ها نام دوستانم را در اینترنت جستجو می کنم تا شاید نشانی از ایشان بیابم ...باورت میشه من هم این کار رو انجام دادم!!! یا چقدر با گوگل ارت ور رفتم تا خونه های قدیمیمون رو پیدا کنم!؟پارسال اونجا بودم ولی هیچ نگذشته دلم تنگ شده

چه میکنه این نیک آهنگ،درددل خیلی از غربت نشینها همین دوراهی است، ماندن و ساختن با غم دوری یا برگشتن و باز ساختن، این بار با محدودیت ها.در کل، اگه ما ایرانی ها در هر کجا از این دنیا که زندگی میکنیم کمی مثل چینی ها یا هندی ها یا ایتالیایی ها و یا حتی یونانی ها متحدتر بودیم، محله ای برای خود داشتیم، آیینها و مراسم متداول داشتیم (و نه چیزی که امروز از نوروز و سیزده بدر و چهارشنبه سوری و و و مانده است.) و کمی یاد میگرفتیم همدیگر را تحمل کنیم، امروز قضیه کاملا متفاوت میشد.

نخندیا!الان اگه بگن ج.ا. ور افتاد و از فردا قراره مثلاً پادشاه سوئد مملکت رو اداره کنه هم من باز برنمیگردم. دلیلش به سادگی این میتونه باشه که تهران زلزله میاد منم اصلاً دلش رو ندارم. پارسال اونجا بودم زلزله شد. شدتش که کم بود ولی از ترس مردن نزدیک بود بمیرم...

ما در همین حومه ونکوور یه شهری داریم به اسم نورت ونکوور که معروفه به محله ایرانیان و پر از ایرانیاست !!!اتفاقا هر سال ۴ شنبه سوری و ۱۳ بدر رو هم به صورت گسترده جشن میگیرن ولی به نظر من فقط به همین دو تا مراسم ختم میشه و بعد از اینها دیگه من ساپورتی نمیبینم!!!سالی ۲-۳ کنسرت ایرانی برگزار میشه که بجای اینکه مردم رو به هم نزدیک کنه شده جای دعوا و لات بازی بعضی از برادران عزیز هموطن !!!

سرما گفتی و کردی کبابم....

دوستان من در ايران هستم و جايتون را خالي ميكنم.زندگي در ايران لذت بخش و شيرين است و من هرگز نميخواهم به خارج از كشور براي اقامت بروم البته سالي يكبار سفر شيرين است.نون ايراني . كوچه هاي تنگ . دعواهاي سر كوچه . محبت دوستان . همدردي همسايه . خنده هاي از ته دل . و ..... هركدامشان به تنهايي براي يك عمر گراني ارزش دارد.بارها گفته ام اينجانب هروقت دلم ميگيرد خانه پدر و مادر . نماز جماعت مسجد . مهماني هزاران قوم و فاميل . تشريفات بريز و بپاش و ....تنها يكي از اين موارد برايم كافيستاز قديم گفته اند مرغ همسايه غاز است . جناب اگر موقعيتي همچون 2 خرداد پيش آمد حتما برگرد كه اگر چند سالي گذشت ديگر نميتواني برگردي.نيك اهنگ جز چند روز زندان كه اينهم دورانش مزايايي داشت از چه قسمت از ايران فرار كرديد ميدانم سخت بود ولي حدث ميزنم تنها لحظه اي از دوران روزنامه جامعه و....را با كل خارج رفتنت عوض نخواهي كرد.ميداني من خارج از كشور را مثل تهران و داخل كشور را مثل روستاهاي ورامين . ... ميبينمممكن است كسي از تهران براي زندگي نتوتند به اين روستا ها برود ولي آرزويش هميشه وجود دارد. تو مجبوري براي كار و كار و كار در تهران بماني ولي هواي تازه و لطافت ييلاق را هرگز فراموش نميكنيدوست عزيز نيك آهنگ برگردبرگردبرگردبرگردبرگردبرگردبرگردبرگردبرگردبرگرد...............................لطفا

وقتی او آمد درهای قلبم را به رويش گشودمو همچون کودکی بی ريا و به دور از تزوير با آغوشی باز پذيرايش شدمغريبه ای را که فرسنگ ها از من دور بود.او قدم به دنيايم گذاشت اما با سنگدلی شاخه های درخت زندگی ام را شکستبی آنکه حتی از نگاه مهربان باغبان شرم کند.پروردگار مهربانم از آسمان نيلگونش نظاره گر بی وفايی هايش بودو صدای شکسته شدن شاخه هايم را می شنيد.اما من و باغبان با محبتم حتی آفتاب و آب چشمه را به روی نا مهربانی هايشنبستيم.به اين اميد که هم رنگمان شود.اما او از جنس ما نبود.او همچون گردباد وزيدشاخه هايم را شکست و شکوفه هايم را پراکند.به اميد آنکه از ما فراتر رود.اما...مثل همه طوفانها مثل همه گردبادهای تلخ آمد.تلخی کرد و رفت.زمستان بود و من صدای قدم هايش را به روی برگهای خشککه دورتر و دورتر می شد شنيدم.او رفت.حالا من و همه نهال ها و بوته ها به امید بهاری ديگرمثل بهاران سال گذشته بار ديگر به اميد شکفتن دوبارهبه انتظار نشسته ايم...اما نه با او... .................به انتظار نشسته ايم...به انتظار نشسته ايم...به انتظار نشسته ايم...به انتظار نشسته ايم...به انتظار نشسته ايم...به انتظار نشسته ايم...به انتظار نشسته ايم...به انتظار نشسته ايم...

آمدم تا مست و مدهوشت كنم اما نشدعاشقانه تكيه بر دوشت كنم اما نشدآمدم تا از سر دلتنگی و دلواپسی گريه تلخی در آغوشت كنم اما نشدآرزو كردم كه يك شب در سراب زندگیچون شراب كهنه ای نوشت كنم اما نشدنازنينم، نازنينم ياد تو هرگز نرفت از خاطرمآمدم تا اين سخن آويزه گوشت كنم اما نشدشعله شد تا به دل خاكستر احساس تو لحظه ای رفتم كه خاموشت كنم اما نشدبعد از آن نامهربانيهای بی حد و فزونسعی كردم تا فراموشت كنم اما نشداما نشداما نشداما نشداما نشداما نشداما نشداما نشداما نشد

خیلی زیبا بوداشک ما داخل نشین ها در اومد

به نظر من وضعیت ایران و خارج رفتن ما مثل این مثال می مونه:مادری رو تصور کنید که یه بچه عقب مونده داره . بعد برای حل مشکل بچه اش رو رها می کنه و میره یه بچه خوشگل و سالم رو به فرزندی قبول می کنه. با فرض اینکه هیچ نگرانی ای از سرنوشت بچه اولش نداشته باشه، قطعا خودش همیشه می دونه این دومی بچه خودش نیست هر چقدر هم به خودش تلقین کنه ببین چقدر خوب و سالمه! یادته اون بچه عقب مونده هه چقدر اذیتت می کرد! اینا فایده نداره. خارج مال ما نیست. ما خارجی حساب می شیم، یعنی متعلق به اینجا نیستیم. من هر چی با خودم کلنجار میرم و هرچی میگم ببین تو اونجایی که مال ایرانی ها نیست باهاشون خیلی بهتر از مملکت خودشون رفتار می شه، باز فایده نداره و تو کتم نمیره. به خاطر همین لحظه شماری می کنم واسه اینکه برگردم تو بغل محمود : )

راست ميگه اين آقا اين بالا... ايرانی ها اصلا با هم متحد نيستند... حالا اين متحد نبودن شون هم بخوره تو سرشون، اصلا يه طوری هستن.. احساس من اينه که از هم فراری اند... اگه تک تک و Idividual باهاشون برخورد کنی می بينی که چقدر آدمهای با کلاس، فهميده و حتی مهربونی هستن، ولی به هم که ميرسن نميتونن با هم بسازن...يه طوری ن..

من تجربه آنچنانی از زندگی تو ايران و ميون ايرانی ها ندارم، هرچی هست تجربيات کودکی و نوجوانی و شيطنت و بازيگوشی و جيغ و داد و بدو بدو هست که اونهم چون تحت ساپرت قوی خانواده بودم توی حساب نيست، ولی وقتی عاقل شدم( ! :) و با ايرانی های اينجا برخورد کردم اين تجربه منه : اونها تو نگاه اول تحويل ات نميگيرن... بعد که حالا تحويل گرفتن يه جوری با پشت چشم نازک کردن نگات ميکنن که انگار اونها صد پله از تو بالاترن و نواده خان قلی خان هستن و تو رعيت و نوکرشون... بعد با دو سه تا سوال کليدی ميخوان سر از کارت دربيارن، و به محض اينکه فهميدن کی هستی و چی هستی رفتارشون تغيير ميکنه و مثلا ميخوان احترام بزارن و جبران کنن و بقيه ماجرا( و بعضی مواقع هم سواستفاده و مزاحمت)... خوب نميشه از همون اول به کسی فخر نفروشيم، احترام بزاريم، ساده باشيم و روابطمون رو اينجوری و معمولی شروع کنيم؟ آيا تو ايران هم همينجوريه؟ اول به آدمها به چشم نوکرشون نگاه می کنن، فخر ميفروشن بعد که فهميدن طرف چکاره اس، تحويلش ميگيرن؟

خلاصه که اگه يه روز من بخوام برگردم ايران مشکل من مشکل سياسی مياسی و کار و پول حتی مشروب و بار و هاليدی و اينجور چيزا نيست، چيزی که منو نگران و منصرف از برگشتن ميکنه زندگی و برخورد با مردم اونجاس... مخصوصا مردمی که تو تهران و شهرهای بزرگ زندگی ميکنن... شنيدم که ميگن اگه برگردی درسته قورتت ميدن..هه..هه..مطمئنا گه يه روز برگردم ميرم تو يه کانتری سايد، يه دهات خوش آب و هوا و دور افتاده، که مردم اش هنوز ساده و بی شيله پيله باشن خونه ميخرم و زندگی ميکنم... يه جائی تو دل کوههای شمال... تو کانتری سايد های اردبيل يا کردستان، شيراز يا حتی جنوب ..
آهان ...راستی... درمورد ايرانی های اينجا.....همه شون هم ( بدون استثنا) در نهايت عاشق ات ميشن و ميخوان باهات ازدواج کنن...هه..هه..همجنس و غيرهمجنس هم تازه گيها ديگه حاليشون نيس :)

آآقا من هم یک مطلبی قبلا نوشته بودم.خیلی دوستاشتم هموطنا بخونن ولی داغ نشد :(اگر دوست داشتید یه سری به ما هم بزنید

کوتاه و بدون شرح بگویم که:علی رغم همه تنفری که از همه چیز ایران دارم دست آخر به این نتیجه رسیدم که بهترین جا برای یک ایرانی همان ایران است...

نه، يک زن کافر در ايران زندگيش زيادی سخت ميشه!

آآقا من هم یک مطلبی قبلا نوشته بودم.خیلی دوستاشتم هموطنا بخونن ولی داغ نشد :(اگر دوست داشتید یه سری به ما هم بزنیدخوندم مطلبتون رو... فکر نمينم واقعيت داشته باشه... البته ممکنه اين شرايط خاص شما باشه، ولی بنظرم عموميت نداره... زندگی هم اينجا به اين تاريکی هم نيست...

شاید ایراد از منه ولی من معنی جملاتی مثل "علی رغم همه تنفری که از همه چیز ایران دارم دست آخر به این نتیجه رسیدم که بهترین جا برای یک ایرانی همان ایران است" رو نمیفهمم. چرا یک جایی که همه چیزش نا خوشاینده بهترین جا برای زندگیه؟ به نظر منم تقریبا همه چیز ایران و فرهنگ ایرانی (غیر از هنرش) ناخوشایند میاد و به همین مناسبت از زندگی در کانادا بی اندازه لذت میبرم. بعد از چند سال سرمایه گذاری عمر در یادگیری زبان و فرهنگ این کشور هر کسی میتونه خودشو عضوی از این کشور بزرگ به حساب بیاره.

Ref: http://balatarin.com/permlink/2008/5/21/1310902

دوپینگ علمی و صدمه به بنیان علم

2008-05-21T00:37:00.004-04:00

تولد رویانا، نخستین جانور همسانه‌سازی شده ...تولد بوآنا، نخستین گاو همسانه‌سازی شده منطقه...دستیابی به فناوری تولید حیوانات تراریخته ...ارائه نخستین نقشه پروتئینی سلول‌های بنیادی جنین انسان ...ترمیم ضایعات نخاعی با استفاده از سلول های شوآن...تولید داروی کنترل ایدز...راه اندازی بزرگترین مجتمع تولید داروهای نوترکیب...تولید داروی اریتروپوئیتین برای درمان بیماران کلیوی
...راه اندازی شرکت تولید کننده بافت مصنوعی انسانی...تولید پوست انسانی برای درمان بیماران سوختگی...

اینها بخشی از ادعاهای یکطرفه و تایید نشده مراجع دولتی یا وابسته به دولت در حوزه علوم زیستی هستند.
این نهادهای تحقیقاتی تولیدی صحت ادعاهای خود را هیچگاه در معرض داوری قرار نداده اند.

برای راستی آزمایی این ادعا ها لازم است پاسخ سوالات زیر را داشته باشیم:

هیئت علمی این مراکز چه کسانی و با چه سطحی از دانش و تجربه هستند؟ سابقه تحصیلات و تحقیقات ایشان چیست؟
مقالات علمی انتشار یافته توسط ایشان در ژورنالهای معتبر بین المللی کدام است؟
این مراکز در حال حاضر در چه زمینه ای بطور تخصصی به خدمت رسانی به بیماران مشغول هستند؟

پاسخ : اعضای هیئت علمی این مراکز در زمینه تخصصی مشابه تا حدی که از اصول ابتدایی دستاوردهای ادعایی اطلاع داشته باشند تحصیل کرده وعلی رغم دانش حرفه ای در زمینه شغلی خود از توان علمی لازم برای به اجرای موثر تکنیک های لازم برای دستیابی به دستاورد ادعایی برخوردار نیستند.
دانشمندان این مراکز هیچگاه نتایج تحقیقات در مراحل مقدماتی و یا پیشرفته را در ژورنالهای معتبر و بعضا حتی ژورنالهای داخلی به چاپ نرسانده اند. مراجعه به مقالات چاپ شده ایشان به خوبی نشان دهنده این نکته است که ایشان عموما دانشمندانی معتبر در زمینه ای دیگر با مبانی نزدیک به دستاوردهای ادعایی هستند!
این مراکزبطور حرفه ای به ارائه خدمات بهداشتی درمانی در زمینه مشابه دیگری مانند درمان نا باروری یا جراحی اعصاب مشغول هستند.

نتیجه: آشنایی مقدماتی دانشمندان با یک سوژه داغ علمی همچون همسان سازی جنین، سلول های بنیادی، نانو تکنولوژی و ... برای سیاست مداران کافی است تا از طریق مدیران عمدتا سیاسی مراکز علمی دانشمندان شاغل در نهاد های دولتی یا وابسته به دولت و نهاد های نظا می را به بزرگ نمایی و داستان سرایی و جعل نتایج تحقیقاتی تشویق کنند. دوپینگ علمی این نهادها ، سایر مراکز علمی و دانشگاهی کشور رابر سر دوراهی تقلید در بزرگنمایی و جعل دستاوردها یا قبول تحقیر و ناتوانی علمی نسبت به مراکز ظاهرا فعال قرار داده است.

راه حل: نهاد نظارتی داخلی و بین المللی؟! نتیجه بررسی ادعای همسان سازی جنین در کره جنوبی یک نمونه از راه برخورد است.

*****
در اینجا برای نمونه به حوزه زیستی بسنده شده است ولی اصول مشابهی در سایر زمینه ها در حال اجرا است. لطفا از ارائه راهکار های خود برای برخورد با این معضل دریغ نفرمایید.

بوش نژاد!

2008-05-18T20:48:00.006-04:00

چشم دل

2008-05-08T21:04:00.004-04:00

وقتی دیدمشون باورش سخت بود که هردو نمی بینند. این دو هیچوقت همدیگه رو ندیدن ، عاشق هم بودن... مرد با دست موهای خانم را نوازش می کرد و صورتشو به پیشونیش می چسبوند تا حسش کنه. .. توصیفش سخته .. . صحنه بی نظیری بود. به معنی واقعی کلمه روشن دل بودن.

برای بزرگ نمایی بیشتر روی تصویر کلیک کنید.

مکان: نیویورک- مشخصات بیشتر تو خود عکس هست!
زمان: دیروز 7 می 2008
عکاس : خودم

جوان ترین پروفسور

2008-05-06T01:09:00.010-04:00

آلیا چهارده ساله
آلیا نوزده ساله

نام و نام خانوادگی :آلیا صبور Alia Sabur

متولد: نیویورک - آمریکا- پاکستانی تبار

تاریخ تولد: 22 فوریه 1989

نام پدر : مارک Mark

نام مادر: جولی Julie

نام دختر عمو: ماهرخ Mahrukh ساکن پاکستان

او در هشت ماهگی خواندن را آغاز کرد و از کلاس چهارم ابتدایی به کالج رفت تا در سطح کارشناسی ریاضیات کاربردی بخواند.

آلیا چهارده ساله بود که مدرک کارشناسی گرفت و دانشجوی دوره کارشناسی ارشد -دکتری دانشگاه درکسل ، نیویورک شد.

او سپس دکتری مهندسی مواد گرفت و جوان ترین فردی بود که وارد دوره فلوشیپ پس از دکتری شد، او نشانگرهای سلولی خاصی را بر اساس تکنولوژی نانولوله ها ابداع کرد که در تحقیقات پزشکی کاربرد دارد.

فقط سه روز مانده بود که نوزده ساله شود که برای تدریس در دانشگاه کنکوک ، سئول ، کره جنوبی پذیرفته شد.

تندروی به چه قیمت

2008-05-04T21:04:00.008-04:00

فرشاد امیر ابراهیمی - عضو فعال انصار حزب ا...- مانور بسیج سال 1989

فرشاد امیر ابراهیمی -فعال حقوق بشر ...- آنکارا 2002

محسن مخملباف -قهرمان انقلابی- پس از ترور نا موفق یکی از عوامل پلیس حکومت شاهنشاهی

محسن مخملباف - هنرمند- کارگردان شناخته شده بین المللی

معصومه ابتکار ملقب به خواهر مری - مترجم گروه تسخیر کننده سفارت آمریکا در تهران

معصومه ابتکار ملقب به قهرمان زمین - در دولت اصلاح طلب محمد خاتمی به درجه های عالی مدیریتی منصوب شد. سابقه عضویت شورای اسلامی شهر تهران. فعال حمایت از محیط زیست.

همه ی انسان ها حق تغییر دارند و حتما از اینکه اسلحه بدستان سابق قلم بدست بگیرن و از عشق، دوستی، پاکی محیط زیست و حقوق بشر دفاع کنند خوشحال میشوم. ولی یک سوال بی پاسخ ذهنم را به درد می آورد: چرا نسل من و شما باید هزینه تندروی ایشان را بپردازد؟

پلیس عشق؟!

2008-05-04T03:01:00.008-04:00

به کدامین گناه؟

و عشق همچنان جاری است ...

حفظ آبرو به چه قیمت

2008-05-01T03:03:00.004-04:00

بی بی سی در یک تلاش رقت بار، در مقدمه نظرخواهی با عنوان حفظ آبرو به چه قیمت؟! در مورد زنای با محارم و زندانی کردن فرزند در اتریش می نویسد: آیا این گونه اتفاقات در جوامع دیگر هم روی می دهد؟ در ایران چطور؟ آیا شما، خود قربانی یا شاهد این اتفاق بوده اید؟
مشکل اینجاست که بی بی سی می خواهد توپ را بیندازد به زمین ایرانی ها. آن هم با این پیام مخفی که" از این چیزها و بدترش هم در ایران هست اما مخفی است."رفتاری بسیار رقت انگیز برای سرپوش گذاشتن روی رسوایی غرب.
اکنون باید از رسانه ی دولتی انگلستان پرسید : حفظ آبرو به چه قیمت؟!

لینک به نظر سنجی وبسایت بی بی سی:

http://newsforums.bbc.co.uk/ws/thread.jspa?sortBy=1&forumID=6059&start=0&tstart=0#paginator

هیلاری! ما فقط دو تا گیلاسیم

2008-04-30T23:21:00.001-04:00

هیلاری! ما فقط دو تا گیلاسیم
ما را نزن! ما را نزن!ما گیلاسیمفقط دو تا گیلاس
گفتی که می‌خواهی ما را از زمین محو کنیگفتی که ما را محو می‌کنیما را محو نکن، ما را نزنما گیلاسیم
هیلاری گفت، می‌خواهم ایران را از صحنه روزگار محو کنمهیلاری!ما را محو نکن، ما را نزنما گناه داریمما فقط دو تا گیلاسیم
هیلاری! وقتی وارد آشپزخانه شدیدر کمد زیر دستشوییسحری است نوشته بر ظرفیدستت را سه بار بر ظرف بکشآیا معجزه‌ای رخ داد؟آیا غولی حاضر شد؟نه، معجزه‌ای رخ نخواهد دادهیچ غولی از بطری وایتکس خارج نمی‌شودحالا درش را دو دور به سمت راست بچرخانمحلول را به آب اضافه کنهمه چیز آماده استآن را روی تاریخ بریزحالا می‌توانی تاریخ را با وایتکس بشویی
این عکس تزیینی است و ربطی به وایتکس ندارد.
تو می‌توانی با محلولی از وایتکسپرچم سرخ ویتنامی‌ها را به پرچم سفید صلح تبدیل کنیو می‌توانی اجساد را از «‌دین بین فو» محو کنیتو می‌توانی مارتین لوتر کینگ سیاه را در وایتکس بیندازیتا از جان وین هم سفید‌تر شودتو می‌توانی افغانستان طالبان را چنان با وایتکس پاک کنیکه پاک‌تر از پاکستان شودتو می‌توانی صدام را با وایتکس از تاریخ عراق محو کنیاما، هنگام پاک‌سازی مواظب باشی جایی منفجر نشود
هیلاری!کمد زیر دستشویی را باز کنتو می‌توانی، تو توانستیتو با همان محلول وایتکس لکه بیل را از دامن مونیکا پاک کردیحتماً با همان وایتکس می‌توانی ایران را هم از صحنه روزگار محو کنیاصولاً وایتکس برای همین استو محو کردن ما کار دشواری نیستما چندین هزار کیلومتر جغرافیاییمو چندین هزار سال تاریخفقط ده دقیقه ما را در وایتکس بخیسانو دکمه قرمز را فشار بده ما محو خواهیم شد
اما نه، دست نگه دارما را محو نکنما را نزن، ما را نزنما گیلاسیمپیش از آغاز دکترین وایتکسشبی پیش از آنوقتی که به باغ رفتی، سری به مغول‌ها بزنبا عرب‌ها ملاقات کنو محمود افغان را ببینمواظب باش!آن‌ها پیش از این به ما تجاوز کردندلکه‌های آن‌ها هنوز روی دامن تاریخ ماستما آن لکه‌ها را نتوانستیم محو کنیمو مغول‌ها و افغان‌ها و عرب‌ها هم ما را محو نکردندتجاوز سختی بود، اما محو نشدیمآن‌ها می‌دانستند که تاریخ با سفره فرق می‌کندتاریخ، سفره نیست که لکه‌اش را محو کنیو سرزمین یک نقشه کاغذی نیستکه پاره‌اش کنی
هیلاری! ای محو کن بزرگای کاش خاطرات خودت را می‌خواندیتا شیوه درست استفاده از وایتکس را فراموش نکنیهیلاری! هیلاری!ما را محو نکن، ما را نزن ما گیلاسیم، فقط دو تا گیلاس
پیش از آن‌که تو تصمیم بگیری ما را محو کنیما سی سال آتش سوزاندیمتا همان یک چهارشنبه هم محو نشودو سی سال سبزه‌ها را گره زدیمتا روی زردمان را کسی نبیندو هشت سال زیر تانک‌ها له شدیمله شدیم، اما محو نشدیمو بیست و پنج سال لگد خوردیمآن‌ها هم می‌خواستند با لگد محومان کنندعقب ماندگی همین است!وایتکس وقتی ندارند، لگد می‌زنندما له شدیم و لگد خوردیم و آتش سوزاندیمهیلاری!لگد خورده‌ها را محو می‌کنی؟
هیلاری!سری به گوگل مپ بزنآیا هرگز فکر کرده‌ای تمام خانه‌هایی که گوگل مپ از بالا نشان می‌دهدخانه‌های آدم‌هاستآدم‌هایی که رویا دارندآدم‌هایی که کابوس می‌بینندآدم‌هایی که نفس می‌کشندآدم‌هایی که عاشق می‌شوندآدم‌هایی که می‌خندندآدم‌هایی که سال‌هاست آرزوهای‌شان رایک روز باد برده استلطفاً پیش از مصرف وایتکس به آدم‌هایی که زیر نقشه زندگی می‌کنند فکر کن
هیلاری!لطفاً ما را محو نکنما یک باغ گیلاسیم
بانوی خندان!می‌دانم که هنوز به آرای نبراسکا و فلوریدا و پنسیلوانیا احتیاج داریاما لطفاً در مصرف وایتکس زیاده روی نکنممکن است آنقدر زیاده‌روی کنیکه مردم آمریکا از وایتکس و محو کردن و هر چیز سفیدی متنفر شوندو اوبامای سیاه را به کاخ سفید بفرستند
ما را نزن! ما را نزن!ما گیلاسیم
نازلی احساس( معصومه مستشار)

Ref: http://radiozamaaneh.com/nabavi/2008/04/post_61.html

The Top 20 Reasons Not to Move to Dubai

2008-04-30T03:51:00.002-04:00

The city of Dubai is located in the northeastern part of the United Arab Emirates (UAE) along the Persian Gulf. Dubai is the capital city of the emirate of Dubai, one of seven emirates forming the United Arab Emirates. Dubai is the second largest city next to Abu Dhabi in UAE. The upper part of the image shows the center of the city, which developed along the west side of the Dubai Creek. Dubai shows 2 different characters: one as a business district lined with high-rise buildings along the creek; and another as a resort with Jumeirah Beach and the safari tour of the desert, shown at the lower left of the image. Off the coast of Jumeirah Beach is the world's largest man-made island resort The Palm in the Persian Gulf, now under construction. The design of the palm tree-shaped island can be seen in the image. The image covers an area of 40.8 x 53.5 km, was acquired 17 September 2003, and is located at 25.2 degrees North latitude, 54.9 degrees East longitude.

Living in Dubai is not wonderful and glamorous, as many would have you believe. Forget about what you’ve read, seen, and heard; those shiny buildings and manmade islands are all just smoke and mirrors. There are so many things wrong with this place that I have decided to compile a list, a must read if you are considering a potential move to Dubai.
1. There is no standard address system making mail-to-the door delivery impossible. In fact, it makes anything nearly impossible. The taxi driver, here for only two days, and having learned English from old Beatles albums has no clue where your house is. He won’t tell you that of course, he’ll just keep calling and saying, “Okay, okay. Yeah, yeah.” When you purchase something that requires delivery they do not have an address line, but a box where you are expected to draw a map. Not able to draw a map? Explain like this: I live on the street after the airport road, but before the roundabout. Go past the mosque and make a U-turn.
2. The government blocks all web sites that it deems “offensive” to the “religious, moral, and cultural values” of the UAE. That’s hard to swallow for a freedom loving American, but I get it. I do not understand, however, why all VOIP access and related web sites are blocked. I guess the government also takes offense to people inexpensively contacting their families back home. You’re welcome to call using the analog service provided by the government-owned telephone monopoly, but it will cost you a whole lot more. So much so, in fact, your frequency of calls will be greatly diminished if you can afford them at all. The government says VOIP is blocked for security reasons, yet even the residents of communist China and North Korea have access to 3. It is really hot outside. Not Florida in July hot; Hot as if you were locked in a car in Florida in July with sufficient humidity to make it feel as though you are drowning. Hot as in 120 degrees with nearly 100% humidity. Do not look to the wind for relief. This is the equivalent of pointing a hairdryer on full blast directly at your face. Pour fine moon dust-like sand over your head as you do this and you get the picture.
4. There are too few trees, plants, and grass – or living things aside from us crazy humans, for that matter. Ever see a bird pant? I have. In my opinion, human beings were not meant to live in such a place. If we were, there would be sufficient water and shade. The only greenery around are the roadside gardens planted by the government, who waters the hell out of them in the middle of the day. Thanks a lot! Didn’t you say we should cut down on our water consumption because you are unable to keep up with the demand? I have an idea: let’s all move someplace where it’s not 120 degrees outside.
5. This country prides itself so much on its glitz and glamour that it put a picture of its 7-star hotel on the license plate. Yet, the public toilets in the king-of-bling Gold Souk district are holes in the ground with no toilet paper or soap. Hoses to rinse your nether regions, however, are provided. This results in a mass of water on the floor that you must stand in to pee. Try squatting without touching anything and keeping your pants from touching anything either. Oh yeah. It’s 120 degrees in there too.
6. This country encourages businesses to hire people from other poor countries to come here and work. They have them sign contracts that are a decade long and then take their passports. Even though taking passports is supposedly illegal, the government knows it happens and does nothing to enforce the law. These poor people are promised a certain pay, but the companies neglect to tell them they will be deducting their cost of living from their paychecks, leaving them virtually penniless – that is, if they choose to pay them. Companies hold back paychecks for months at a time. When the workers strike as a result, they are jailed. Protesting is illegal, you see (apparently this law IS enforced).
These people will never make enough to buy a ticket home and even if they do, they do not have their passports. They live crammed in portables with tons of others, in highly unsanitary conditions. The kicker: they are building hotels that cost more to stay in for one night than they will make in an entire year. Things are so bad that a number of laborers are willing to throw themselves in front of cars because their death would bring their family affluence in the form of diya, blood money paid to the victim’s family as mandated by the government.
7. Things are not cheaper here. I’m sick of people saying that. I read the letters to the editor page of the paper and people say to those who complain about the cost of living rising here, “Well, it’s cheaper than your home country or you wouldn’t be here.” The only thing cheaper here is labor. Yes, you can have a maid – but a bag of washed lettuce will cost you almost $10.
8. There are traffic cameras everywhere. I consider this cheating. Where are the damn cops? I drove around this city for weeks before I ever even saw a cop. Trust me, they need traffic cops here. People drive like idiots. It’s perfectly okay to turn left from the far right lane, but speeding even just a couple of kilometers over will get you fined. These cameras are placed strategically as you come down hills, or just as the speed limit changes. Before you know it…BAM! Fined. Forget to pay the bill and your car will be impounded..
9. The clothing some of these women wear makes no sense to me. I understand that as part of your religion you are required to dress in a particular way, but a black robe over your jeans and turtleneck and cover your head when it is 120 degrees outside? In the gym some women wear five layers of clothing…sweatpants and t-shits over sweaters with headscarves. Yet the men’s clothing makes absolute sense: white, airy, and nothing underneath but their skivvies.
10. People stare at you. I am sick of being stared at. I’m stared at by men who have never seen a fair-skinned blue-eyed woman before, or who have and think we are all prostitutes so it’s okay to stare. They stare at me when I am fully covered or with my husband, and even follow me around. It’s beyond creepy and has brought me to tears on more than one occasion. The staring is not limited to men, either. I’m stared at angrily by female prostitutes who think I am running in on their territory by having a few drinks with my husband at the bar. 11. Prostitutes? Oh hell yes, there are prostitutes. Tons of them. So, let me get this straight, I can’t look at a naked picture of a person on the Internet in the privacy of my home, but it is okay to go out in public and buy a few for the night?
12. Alcohol can only be sold in hotels and a handful of private clubs. A person must own a liquor license to consume in the privacy of their own home. To obtain a liquor license you must get signed approval from your boss, prove a certain level of salary that determines how much you are allowed to buy, and then submit several mug shots (aka passport photos) for approval. Pay the fee and the additional 30% tax on every purchase and you may drink at home. Then again, you can just pick up a few bottles in the airport duty free on your way in to the country, but two is the max. Why not just drive out to Ajman where it’s a free-for-all and load up the SUV? It’s easy enough, but crossing the Emirates with alcohol is illegal – particularly in the dry emirate of Sharjah, which just happens to lie between Dubai and Ajman. Go figure.
13. Not only do you have to get your boss’s approval to obtain a liquor license, but you must also get the company’s approval to rent property, have a telephone, or get satellite TV.
14. Back to the craziness on the roads: If I see one more kid standing up and waving to me out the back window while flying down the road at 160 kph…whatever happened to seatbelts?
15. When is the weekend again? Let me get this straight: the weekend used to be Thursday and Friday, but no one took off all of Thursday, just a half day really. Now the government says Friday and Saturday are the weekend, but some people only take off Friday, others still take a half day on Thursday, but some might just take a half day on Saturday instead. Anyway you slice it, Sundays are workdays and little business can be accomplished Thursday through Saturday.
16. There are few satellite television operators:. The movie channels play movies that are old and outdated. Many of them went straight to video back in the States. Every sitcom that failed in the US has been purchased and is played here. Old episodes of Knight Rider are advertised like it is the coolest thing since sliced bread. The TV commercials are repeated so often that I am determined NOT to buy anything I see advertised on television here just for thee principle of it. When I say repeated often, I mean every commercial break - sometimes more than once.
17. The roads are horribly designed. Driving ten minutes out of the way to make a U-turn is not uncommon. People are not able to give directions most of the time (remember reason #1), and the maps are little help because most have few road names on them, if any. Where is interchange four? You just have to hope you got on the freeway in the right place and start counting because they are not numbered. Miss it and you’ll likely end up on the other side of town before you are able to turn around and go back.
18. Taxi drivers are dangerous and smell. Taxi drivers work very hard here to earn a living because travel by taxi is still relatively inexpensive, even though the cost of living is not (see reason #7). Because of this you may have a driver who has had little sleep or the opportunity to shower for several days. Many of these drivers have just as much difficulty finding their way around as you do, but add to this a third-world country driving style and extreme exhaustion and, well, remember to buckle up for safety.
19. Speeding is an Emirati sport and Emirates Road is just an extension of the Dubai Autodrome. I know I keep mentioning the roads, but really, much of this city’s issues are encompassed by the erratic and irrational behavior displayed on its streets. Visions of flashing lights on even flashier, limo-tinted SUVs haunt me as I merge on to the highway. Local nationals are somehow able to get the sun-protecting dark window tint denied to us lowly expats and use it to hide their faces as they tailgate you incessantly at unbelievably high speeds, their lights flickering on and off and horn blaring repeatedly. It doesn’t matter that you can’t get over, or if doing so would be particularly dangerous, they will run you off the road to get in front of you. Don’t even think about giving someone the finger; the offense could land you in jail. Tailgating is, unbelievably, legal.
20. Dubai is far from environmentally friendly. Ever wonder how much damage those manmade islands are doing to the delicate ocean ecosystem? Coral reefs, sea grasses, and oyster beds that were once part of protected marine lands lie choked under a barrage of dredged up sea sand. Consider the waste that occurs from erecting buildings on top of these sand monsters and from the people that occupy them coupled with the lack of an effective recycling program and you have an environmental disaster on your hands. Add to this more gas guzzling SUVs than fuel-efficient cars on the road and the need for 24-hour powerful air-conditioning and its evident that the environment is not high on the priority list of the UAE.
So while I’m sure there are benefits to living in Dubai, tax breaks, multi-cultural environments, and beautiful buildings aside, reconsider your plans to move here if any of the above mentioned reasons strikes a chord within you. Dubai is a city caught in an identity crisis. Struggling somewhere between its desire to be a playground for the rich and its adherence to traditional Islamic roots, rests a city that lacks sufficient infrastructure to support its delusions of grandeur. Visit if you must, but leave quickly before you are sucked into its calamitous void.these inexpensive calls.

Persian Gulf

2008-04-30T03:42:00.003-04:00

The name Persia has always been used to describe the nation of Iran, its people, and its ancient empires since 600 BC. It is derived from the ancient Greek name for Iran's maritime province, called Fars or Pars in modern Persian, Pars in Middle Persian and Pârsa in Old Persian, a word meaning "above reproach." Persis is the Hellenized form of Pars, and through the Latinized word Persia, the other European nations came to use this word for the region. This area was the core of the original Persian Empire.
Since ancient times almost all foreigners referred to the entire country as Persia until March 21, 1935, when Reza Shah Pahlavi formally asked the international community to call the country Iran — a name that the people of Persia, themselves, used to refer to their country since the Sassanian period. "Iran" means "Land of Aryans". In 1959 some Persian scholars protested to the government that the name change had separated the country from its ancient civilization. Therefore, the late King Mohammad Reza Shah Pahlavi announced that both Persia and Iran can be used in Western languages.
Without disparaging the Arabs, Iranians are proud of their non-Arab heritage and strongly resent any attempt at denigrating or changing any aspect of their Iranian heritage. And the Persian Gulf occupies a pivotal place in the Iranian history and culture. Furthermore, Iran abuts the Persian Gulf for 2,000 Km, while about a dozen recently-created Arab Sheikhdoms and emirates border the Gulf on the other side.
The historical and geographical name of the Persian Gulf has been endorsed and clarified by the United Nations on many occasions and is in use by the UN, its member states, and all other international agencies worldwide. The last UN Directive confirming the name of the Persian Gulf was (reference ST/CS/SER.A/29/Add.2) on August, 18th 1994.
The worldwide Iranian people are deeply affronted by this arrogant designation of "The Arabian Gulf." We demand, in the strongest possible terms, that you take immediate steps to restore the rightful name of the Persian Gulf to the waterway on your TV programms (including Megastructures in which you used the fake name for Persian Gulf) and in your website.

UFO Dogfight over Tehran

2008-04-21T04:25:00.001-04:00

Declassified Defense Intelligence Agency documents released under the Freedom of Information Act reveal that on 19 September, 1976, an unusual incident occurred over Tehran, Iran. At about 0030 hours, the Imperial Iranian Air Force command post at Tehran received four telephone reports from citizens in the Shemiran (north of Tehran). Some of the callers reported seeing a bird-like object in the sky, while others reported a helicopter with a bright light. When the command post found that there were no helicopters airborne at that time, they called General Yousefi, assistant deputy commander of operations. General Yousefi at first said the object was only a star, but after talking to the tower at Mehrabad Airport, he looked for himself and saw a very bright object larger than a star. At that point he decided to scramble one F-4 Phantom jet from Shahrokhi Air Force Base in Hamadan. At 0130 hours, the F-4 took off and proceeded to a point 40 nautical miles north of Tehran. It was noted that the object was of such brilliance that it could be seen up to 70 miles away. When the F-4 came to within about 25 nautical miles of the object, the jet suddenly lost all instrumentation and communications. The pilot broke off the intercept and turned away. When the F-4 had turned back toward Shahrokhi, the aircraft regained instrumentation and communication. At 0140 hours, second F-4 was scrambled, piloted by Lieutenant Jafari and it acquired a radar lock on the object at 27 nautical miles range. The radar signature of the UFO resembled to that of Boeing 707 aircraft. Closing on the object at 150 nautical miles per hour. At a range of 25 nautical miles, the object began to move, keeping a steady distance of 25 nautical miles from the F-4.The size of the object was difficult to determine due to its intense brilliance. The lights of the object were alternating blue, green, red, and orange, and were arranged in a square pattern. The lights flashed in sequence, but the flashing was so rapid that they all could be seen at once.
As the object and the F-4 continued on a southerly path, a smaller second object detached itself from the first and advanced on the F-4 at a high rate of speed. Lieutenant Jafari thinking to be under attack, he launch an AIM-9 sidewinder missile, but he suddenly lost all instrumentation, including weapons control, and all communication. The F-4 pilot then instituted a turn and a negative G dive as evasive action. The object fell in behind him at about 3 to 4 nautical miles distance for a short time, then turned and rejoined the primary object.Once again, as soon as the F-4 had turned away, instrumentation and communications were regained. The F-4 crew then saw another brightly lit object detach itself from the other side of the primary object and drop straight down at a high rate of speed. The F-4 crew expected it to impact the ground and explode, but it came to rest gently. The F-4 crew then overflew the site at a decreased altitude and marked the position of the light's touchdown. Then they landed at Mehrabad, noting that each time they passed through a magnetic bearing of 150 degrees from Mehrabad, they experienced interference and experienced communications failure. A civilian airliner that was approaching Mehrabad experienced a loss of communications at the same position relative to Mehrabad. As the F-4 was on final approach, they sighted yet another object, cylinder-shaped, with bright, steady lights on each end and a flashing light in the middle. Mehrabad tower reported no other aircraft in the area, but tower personnel were able to see the object when given direction by the F-4 pilot. The next day, the F-4 crew flew out in a helicopter to the site where they had seen the smaller object land. In the daylight, it was determined to be a dry lake bed, but no traces could be seen. They then circled the area to the west and picked up a noticeable "beeper" signal. The signal was loudest near a small house, so they landed and questioned the occupants of the house about any unusual events of the previous night. They reported a loud noise and a bright light like lightning.Further investigation of the landing site, including radiation testing of the area was apparently done, but the results were never made public. Since this event occurred before the fall of the Shah, any records in Tehran itself may be lost.
The Defense Intelligence Agency itself called this report:An outstanding report. This case is a classic which meets all the criteria necessary for a valid study of the UFO phenomenon...
One of the most interesting things about this event is that it was apparently detected by a military spy satellite. This satellite, the DSP-1, was launched to warn of ballistic missile launches by detecting infrared heat sources. It was used to detect SCUD missile launches during Desert Storm. An analysis of computer printouts from DSP-1 by researchers Lee Graham and Ron Reghr, of Aero-Jet in California, shows that it definitely detected an infrared anomaly over Tehran at the time of the UFO event reported above.

قهرمانان نیروی هوایی

2008-04-21T03:51:00.005-04:00

Iranian POW pilots in Iraq Iranian POW pilots in Iraq

List of IIAF Pilots who were killed in the Iran/Iraq War
Not included:Pilots who ejected and became POWPilots who ejected and were rescued

This List Is NOt Complete,

1.....……….. Abbaszadeh

2. Khosrow Abdolkarimi ( F-4 ) Bandar Abbas

3. Abolhassan Aboulhassani ( F-5 ) over Ducan Dam

4. . ...........Aboutalebi ( F-4 ) Bushehr

5. Hassan Afshin Azar ( F-5 ) Mossel

6. Nosratolah Aghaee ( C-130 ) Kermanshah 17-Dey -1359 =

7 Jan. 19817. Abbas .Akbari ( F-4 ) Iraq

8. Khosrow Akhbari ( F-4 )

9. Davood Akradi ( F-4 ) Karkook 8 - Aban - 1360 = 30 Oct.1981

10. Hashem Ale Agha ( F-14 )Soth of Mahshahr 20 - Mordad - 1363 =

11 Aug. 198411.Ali AliAkbaree ( F-4 ) Bushehr 7- Mehr - 1359 = 29 Sept. 1980

12. Masoud Amiri ( F-4 ) Aban - 1359 = Oct 1980

13. Asdolah AssadZadeh ( F-5 ) Over Faw

14. Asadolah Barbari ( F-5 ) over Rawanduz Mehr - 1359 = Sept 1980

15. Mohammad Balazadeh ( F-5 )

16. ............... Bazargan ( F-5 )

17. Ebrahim Dalal Khosh 1363= 1984

18. Mohammad Hossein Darabee ( F-5 ) 26 - Esfand - 1359 = 17 Mar. 1981

19. Ebrahim Delhamed ( F-5 ) 70 NM from Mosul

20. Masihollah Din Mohammadi ( F-4 )

21. Abbas Dooran ( F-4 ) Baghdad 31- Tir - 1361 =

22 Jul. 198222. Ali Eqhbali ( F-5 ) over Aqrah

23. Mohammad Eshghipoor ( F-4 )

24. Abbas Eslami Nia ( F-4 ) Baghdad 1359 = 1980

25. ...............Farahani ( F-4 ) Ghasre Shirin

26. Abbas Fazilat ( F-5 ) over Rawanduz

27. Hassan Ghahestani ( F-4 )

28. Reza Gharabaghi ( F-4 ) ????

29. Gholam Gholamrezaee ( F-5 ) Karkheh - Dezful 1359 = 1980

30. Khalil Ghobadi ( Helicopter ) Iraq

31. Mansour Ghoreyshi ( F-4 ) Mahshahr 18 - Bahman - 1359 = 7 Feb. 1981

32. Bijan Haji ( F-4 ) Over Iraq 18-Mehr 1359 = 10 Oct. 1980

33. Bijan Harooni ( F-5 ) Dezful

34. .......... Hassani ( F-4 ) Ghasre Shirin (Back Seat of Khoojasteh Nikoo) 1359 = 1980

35. Alireza Hashemian

36. ....... …. Heidarian ( F- 4 ) Back seat of Salehi

37. Homayoun Hekmati ( F-4 ) Over Persian Gulf 15-Khoordad 1363 = 5 June 1984

38. Behzad Hessaree ( F-4 ) Bushehr 9 - Mordad - 1361= 31 Jul.1982

39. Ali Ilkhani ( F-4 ) Ghasre Shirin 1359 = 1980

40. Ali Jahan Shahloo ( F-5 ) Iraq

41. Ghafoor Jeddi ( F-4 )

42. ...............Jooraki ( F-5 ) West of Dezful after Take off 13 - Mehr - 1359 = 5 Oct. 1980

43. ................Kadkhoodaee ( F-4 ) Over Mahshahr 6 - Mehr - 1359 = 28 Sept. 1980

44. Mohammad Kambakhsh Ziaee ( F-5 ) 3 - Azar - 1359 = 24 Nov. 1980

45. Mohammad Reza Karimi ( F-4 ) Over Mahshahr 6 - Mehr - 1359 =28 Sept.1980

46. Parviz Keyhani Nejad ( F-4 ) Over Persian Gulf

47. ...................................... ( F-4 ) Ghasre Shirin 1359 = 1980

48. Youness Khoshbin ( F-5 ) Karkheh - Dezful

48. Gholamhossein.Khoshniyyat ( F-5 ) Karkheh - Dezful

50. Ali Khosravi ( F-4 )

51. Ghodrat Kianjoo ( F-4 ) Back seat of Bijan Haji

52. Mansour Mohammadi Azad( F-5 ) Iraq - Esfand 1365 = Feb 1987

53. Masoud Mohammadi ( F-4 ) Bushehr 10 - Mehr - 1359 = 2 Oct. 1980

54. Darioush Nadimi ( F-4 )

55. ................. Nadernia ( F-5 ) 7 - Bahman - 1361 = 27 Jan. 1983

56. Ali Naghdi ( F-5 ) over Sahand & Sabalan-Tabriz

57. Reza Nooroozi ( F-4 ) West of Ahwaz

58. Mohamad Ali Oshrieh ( Farzin ) ( F-5 ) Over Ahwaz

59. Firooz Rahmatian ( F-4 )

60. Gholamreza Ranjbaran ( F-5 ) 15 - Mehr - 1359 = 7 Oct. 1980

61. ……… Roozitalab ( F-4 )

62. . ……… Salehi ( F-4 ) (Pour Rezaee's Wingman on 28 - Shahrivar - 1359 = 19 Sept.1980)

63. Mostafa "Hamid" Saghiri ( F-4 ) 3 - Mehr - 1359 = 25 Sept.1980

64. Changiz Sepehr ( F-5 ) West of Dezful after Take off 13 - Mehr - 1359 = 5 Oct. 1980

65. Mohammad Shademan Bakht ( F-4 ) 2 - Aban - 1359 = 24 Oct. 1981

66. Ali Shamsbeigi ( F-4 ) was killed on Helicopter crash over Kurdestan

67. Homayoon Shoghi ( F-4 )

68. Mohammad Shokoohnia ( RF-4 )

69. Ali Soleimani ( F-4 ) Persian Gulf

70. Shahab Soltani ( F-5 ) West of Dezful

71. Abbas Soltani ( RF-4 )

72. Hassan Taleb Mehr ( F-4 ) Ghasre Shirin 1359 = 1980

73. Ebrahim Tavakoli ( F-5 ) over Ahwaz

74. .............................. ( F-4 ) Paveh 1358 =1980

75. Mahmood Yazdanpanah ( F-5 )

76. Parviz Zabihi ( F-5) NE of Mosel 22 - Aban - 1359 = 13 Nov. 1980

77............. Zanjani ( F-5 ) Soleymanieh (Sharifi's Wingman)

78. Kazem Zarifkhadem ( F-5 ) West of Khaneh inside Iraq 3 - Mehr - 1359 = 25 Sept. 1980

79. Fereidoun Zolfaghari ( RF-4 ) Over Majnoon Islands 20 - Khoordad - 1359 =10 June 1980

80. Mehdi Zooghi ( F-4 ) Iraq

81. Rahim Zooghi Moghadam ( F-5 ) 21- Tir - 1361= 12 Jul. 1982
to be completed ...

مجوز استفاده از رادیو در منزل

2008-04-21T03:34:00.003-04:00

مجوز نصب یکدستگاه رادیو کنسرت در منزلی در چهار راخ مختاری تهران 1319

ظاهرا این هفتاد سال اخیر اوضاع خرابتر شده چون دستکم اون موقع برا رادیو مجوز می دادن ولی حالا برا ماهواره مجوز هم نمیدن

آمُل ۱۳۳۷ خورشیدی.

2008-04-19T03:36:00.002-04:00

رسانه، خلیج فارس و شاهنامه

2008-04-18T00:26:00.003-04:00

کانال نشنال جیوگرافی یه برنامه داره به اسم مگا استراکچرز تو این برنامه در مورد ساختمان ها پل های عظیم یا هر ساخته دست بشر که دارای عظمت از لحاظ اندازه یا تکنولوژی و.... گزارش می ده. توی یکی از این برنامه ها به زیبایی تمام نحوه ساخت برج العرب در دوبی نمایش داده می شه و بارها نام جعلی خلیج عر ب ی بکار بیان میشه و بطور مکرر از تیزهوشی و درایت و مدیریت شیخ فلان (یادم نیست اسمش چی بود) یاد می شه ، طوری که فکر می کنی این برنامه داره از تلویزیون دبی پخش می شه. لازم به ذکر که این برنامه در کشور های مختلف از انگلستان گرفته تا هند و ... عیره پخش میشه واعراب از این طریق با صرف هزینه ای کم به نسبت دستاوردی که براشون داره باره نام جعلی رو مطرح و برای مصطلح کردنش تلاش می کنند

کانال هیستوری برنامه ای داره به نام مهندسی امپراتوریها که هر بار به بررسی یکی از امپراتوری های گذشته می پردازه، یکی

از اونا در مورد امپراتوری پارس است

کانال نیکلودین برنامه ای داره به نام اوتار : اوتار یه کودک افسانیه که صدو خورده ای سال سنشه! و در گیر ماجراهای افسانه ای بسیاری می شه و کل داستان اشاره به چهار عنصر آب ، خاک، آتش و هوا و تسلط اقوام و ملل گوناگون به این عناصر است و ظاهرا اوتار تنها کسی است که به هر چهار عنصر تسلط داره و قراره در جنگ با پلیدی جهان رو از نابودی نجات بده. کلیت داستان همان جنگ همیشگی و باستانی خیر و شره و چارچوب داستان بسیار شبیه داستانهای باستانی خود مونه اما در قالبی بسیار گیرا و مدرن

عرب ها در کنار سرمایه گزاریشون روی ساخت ساختمانهای مدرن و جزایر مصنوعی در آن بیابان برهوت با سرمایه گزاری و حمایت مالی از نشنال جیوگرافی و سایر رسانه ها براحتی دست به تبلیغات غیر مستقیم، عظیم و در عین حال زیبا و مردم پسندی زدن که در مقابل بی کفایتی، انفعال و دشمن تراشی مدیران و سرمایه گزاران دولتی و خصوصی ما مسلما برد با آنها خواهد بود. مگر آنکه راه دیگری جز اکتفا به ایمیل و امضا برای تثبیت حق خود پیدا کنیم.
هیستوری چنل در برنامه مهندسی امپراتوری تلاش کرد به امپراتوری پرافتخار پارس هم بپردازد ولی مقایسه ای ساده با سایر برنامه ها در مورد امپراتوری روم و مصر باستان نشان دهنده محدودیتهای فراوان تیم تهیه کننده بود. آیا برای سازمان های دولتی و یا حتی خصوصی ایران امکان حمایت علمی و بویژه مالی از آن وجود نداشت؟

آیا برای گروه های هنری و استعدادهای سینمایی و انیمیشن ایران این امکان وجود ندارد که برای یکبار هم که شده با دیدی مدرن ، داستانها و اسطوره های نا همچون شاهنما را به تصویر در بیاورند؟ ایا نمی توان رستم را برای یکبار هم که شده بدون یال و کوپال عتیقه در قالب یک قهرمان مدرن با الهام از دیجیمون و اوتار یا ترانسفورمرز به تصویر درآوریم و رخش را همیشه اسبی بزرگ نبینیم؟ اگر توان علمی یا استعداد اجرایی داخلی نداریم ، آیا نهاد های دولتی و خصوصی ما با صرف هزینه امکان سفارش تهیه یک انیمیشن مدرن و زیبا بر اساس افسانه های بسیار این آب و خام ندارند؟ استفاده هوشمندانه از رسانه های قدرتمند غربی با حمایت مالی از آنان نقشی بسیار فراتر از تصور در تامین امنیت ملی و تثبیت حقوق ایران و ایرانی دارد.

آیا می دانید بسیاری از غربی ها نمی دانند امپراتوری پارس همان امپراتوری ایرانیان است؟این نشانه ناتوانی ما در شناساندن خود است. آیا می دانید یک آمریکایی امنیت خود و کودکش را از جان هزاران ها انسان مثلا ایرانی یا عراقی و.... مهمتر می داند. به او حق بدهید! آیا ایران امکان حمایت مالی از رسانه های غربی و یارگیری به نفع خود را ندارد؟

بیایید تا دیر نشده به جای امضا جمع کردن برای گوگل و نشنال جیوگرافی برای احمدی نژاد و زرغامی و سرمایه گزاران ایرانی امضا جمع کنیم

لحظه ای فرخنده از تاریخ ایران

2008-04-07T04:33:00.002-04:00

تیمسار دریابد رسایی در جزیره ابوموسی پس از آزاد سازی آن توسط نیروی دریایی شاهنشاهی ایران.
نهم آذر ۱۳۵۰ خورشیدی- ۳۰ سپتامبر ۱۹۷۱

هدف آمريكا از برنامه دفاع موشكي

2008-04-03T04:11:00.003-04:00

رئیس جمهور آمریکا در توجیه برنامه گسترش سپر دفاع ضد موشکی به شرق اروپا گفته است که این برنامه برای مقابله با تهدیدات موشکی کشورهایی مانند ایران ضرورت دارد.

ولي:

منحنی مسیر یک موشک بالستیک قاره پیما را می توان به چهار مرحله تقسیم کرد. اولین مرحله، فاز پرتاب است که ۲۰۰ تا ۳۰۰ کیلومتر اول حرکت موشک را در بر می گیرد.
این فاز در موشک های با سوخت جامد تا ۳ دقیقه و در موشک هایی با سوخت مایع تا ۵ دقیقه طول می کشد. موشک در پایان این مرحله شامل کلاهک هسته ای، سیستم کنترل، موتورهای کوچک و وسایلی است که برای کمک به نفوذ در سیستم دفاعی دشمن طراحی شده اند. صدها هزار قطعه کوچک تولید پارازیت در موشک جاسازی شده اند تا سیستم راداری دشمن مختل شوند.
در فاز دوم که توسط سیستم کنترل برنامه ریزی می شود کلاهک ها تک تک به سمت هدف پرتاب می شوند. هر موشک عموما میان ۳۰ تا ۴۰ کلاهک حمل می کند.
در فاز سوم موشک که تمامی کلاهک های واقعی و قلابی خود را پرتاب کرده، در ارتفاع ۱۲۰۰ کیلومتری به حرکت ادامه می دهد. زمان این مرحله بین ۱۵ تا ۲۰ دقیقه است.
فاز چهارم، آخرین و کوتاه ترین مرحله است و کمتر از یک دقیقه طول می کشد. بقایای موشک با سرعت ۷کیلومتر بر ثانیه وارد جو می شوند و بالاخره از بین می روند.
بهترین زمان برای هدف قرار دادن یک موشک بالستیک همان مرحله اول یا فاز پرتاب است. اما این تنها در صورتی رخ می دهد که سیستم دفاع موشکی در کمتر از ۵۰۰ کیلومتری محل پرتاب موشک با سوخت مایع (یا ۳۰۰ کیلومتری موشک با سوخت جامد) مستقر شده باشد. ولي سيستم دفاع موشكي آمريكا در فاصله بسيار بيشتري از ايران مستقر خواهد شد. چرا كه هدف روسيه است نه ايران!

Ref.: http://www.bbc.co.uk/persian/worldnews/story/2007/06/070613_an-missile-system.shtml

اطلاعیه ای برای پاکیزگی!

2008-04-03T03:47:00.003-04:00

یک اطلاعیه خنده دار و در عین حال هوشمندانه نصب شده در دستشویی برا حفظ پاکیزگی!

اشتباه در شبیه سازی!

2008-04-01T02:49:00.000-04:00

وقتی که دانشمندها در کلونینگ اشتباه می کنند

ابن سيرين

2008-04-01T02:21:00.001-04:00

نامش محمد فرزند سيرين بصرى است، در تعبير خواب قدرت فوق العاده اى داشت ، سرچشمه تعبيرش ذوق سالم و فكر نافذ او بود .
در تطبيق خواب با حقايق انسان عجيبى بود ، براى تعبير خواب از لطايف قرآن و روايات استفاده مى كرد .
نوشته اند مردى از او پرسيد : تعبير اذان گفتن در عالم خواب چيست ؟ گفت : رفتن به حج . ديگرى همين مسأله را پرسيد ، گفت : دست به دزدى برده اى . آنگاه درباره اختلاف اين دو تعبير با اينكه خواب هر دو يكى بود گفت : چهره اولى را چهره اى نيكو و پسنديده و دينى ديدم ،
تعبير خوابش را از آيه ( وَاَذِّنْ بِالنَّاسِ بِالْحَجِّ ) گرفتم . اما چهره دومى را چهره خوبى نديدم ،
تعبير خوابش را از آيه ( اَذَّنَ مُؤَذِّنٌ اَيَّتُهَا الْعيرُ اِنَّكُمْ لَسَارِقُونَ ) گرفتم .
ابن سيرين مى گويد : در بازار به شغل بزازى اشتغال داشتم ، زنى زيبا براى خريد به مغازه ام آمد ، در حالى كه نمى دانستم به خاطر جوانى و زيباييم عاشق من است ، مقدارى پارچه از من خريد و در ميان بغچه پيچيد ، ناگهان گفت : اى مرد بزاز ! فراموش كرده ام پول همراه خود بياورم ، اين بغچه را به كمك من تا منزل من بياور و آنجا پولش را دريافت كن ! من به ناچار تا كنار خانه او رفتم ، مرا به دهليز خانه خواست ، چون قدم در آنجا گذاشتم در را بست و پوشش از جمال خود برگرفت و اظهار كرد : مدتى است شيفته جمال توام و راه رسيدن به وصالت را در اين طريق ديدم ، اكنون در اين خانه تويى و من ، بايد كام مرا برآورى ، ورنه كارت را به رسوايى مى كشم .
به او گفتم : از خدا بترس ، دامن به زنا آلوده مكن ، زنا از گناهان كبيره و موجب ورود به آتش جهنم است . نصيحتم فايده نكرد ، موعظه ام اثر نبخشيد ، از او خواستم از رفتن من به دستشويى مانع نشود ، به خيال اينكه قضاى حاجت دارم مرا آزاد گذاشت . به دستشويى رفتم ، براى حفظ ايمان و آخرت و كرامت انسانى ام سراپاى خود را به نجاست آلوده كردم ، چون با آن وضع از آن محل بيرون آمدم ، درب منزل را گشود و مرا بيرون كرد ، خود را به آب رساندم ، بدن و لباسم را شستم ، در عوض اينكه به خاطر دينم خود را ساعتى به بوى بد آلودم ، خداوند بويم را همچون بوى عطر قرار داد و دانش تعبير خواب را به من مرحمت فرمود

Ref.: http://psaboori.blogfa.com/post-21.aspx

NEW YORK

2008-03-30T03:54:00.005-04:00

Under construction!

نیروی هوایی در جنگ ایران و عراق

2008-03-29T03:17:00.003-04:00

22 سپتامبر ۱۹۸۰ برابر با 31 شهریور ۱۳۵۹ عراق جنگی علیه ایران آغاز کرد که ۸ سال به طول انجامید و پس از جنگ ویتنام، طولانی‌ترین جنگ قرن اخیر بود. اگرچه این جنگ، از نظر تداوم و شدت، بیشتر نبردی زمینی بود اما نیروهای هوایی نیز نقش قابل توجهی را ایفا کردند. پس از حملهء ناشیانهء هواپیماهای عراقی در روز اول جنگ برای از صف خارج کردن نیروی هوایی ایران، این نیرو توانست با وجود بروز مشکلات ناشی از غافلگیری و پیامدهای انقلاب نظیر تصفیهء ارتش از افسران زبده ولی وفادار به حکومت پیشین، به سرعت توانایی خود را بازیابد و با اجرای عملیات شجاعانه و حتا عجیب، ۱۳ لشگر آفندی عراق را در وضعیت مشکلی قراردهد. نیروی هوایی ایران در بین نیروهای منطقه در آن زمان، به دلیل توجه، خریدهای هوشمند و آموزشهای باکیفیت انجام شده در دوران پادشاه فقید ایران، بی‌شک برترین بود. براساس آموزشهای داده شده به این نیرو، تخصص آن کندکردن و حتی توقف واحدهای سنگین زرهی (حتی اگر مربوط به ارتش سرخ باشد) بود. بی‌شک به جز برخی مقاومتهای کوچک مردمی در روزهای اول جنگ، این نیروی هوایی و هوانیروز ایران بود که نوک پیکان مهاجمان را کند کرد. در زمانی که هنوز نیروهای کلاسیک ارتش و نیروهای داوطلب مردمی فرصت استقرار و سازماندهی را در مرزهای غربی و جنوبی نیافتند، نیروهای هلیکوپتری و هوایی ایران ۱۵ روز حیاتی را از ارتش عراق گرفتند و آنها را در حوالی مرز ایران، زمین‌گیر کردند. نیروی هوایی ایران هزاران سورتی پرواز بر روی ارتش عراق اجرا کرد و هلیکوپترهای کبرای ایران با موشکهای ضدتانک تاو و راکتهای 70 میلی‌متری خود، جهنمی از آتش را برای واحدهای زرهی عراق ایجاد کردند. نیروی هوایی با زدن عقبه‌ها، واحدهای تدارکاتی و حتی باندهای پروازی نیروی هوایی عراق، شتاب ارتش بعث را به شدت کاهش داد، به نحوی که 4 تیپ ویژهء عراق، 35 روز حیاتی را در پشت دروازه‌های شهر کوچک خرمشهر از دست دادند و 2 لشگر مکانیزهء عراق در پشت دروازه‌های اهواز مجبور به عقب‌نشینی گشتند. جنگنده‌های F-4E نیروی هوایی ایران با بهره‌گیری از خلبانانی که به قول پادشاه فقید ایران، بیش از ارزش هواپیمایشان، صرف آموزش آنها گشته بود، با موشکهای ماوریک و بمب‌های سقوط آزاد Mk 82 پیشروی سریع ارتش بعث عراق را کامل متوقف کردند، به نحوی که ارتش بعث، نتوانست حتا شهرهای مرزی نظیر آبادان، ایلام، اهواز، کرمانشاه را به تصرف خود درآورد، زیرا صدام با توجه به شرائط نامساعد نیروهای ارتش ایران، گمان می‌برد ظرف یک هفته به تهران برسد! نبرد در دریا نیز به طور کامل با پشتیبانی جنگنده‌های F-14A و F-4E نیروی هوایی ایران، به طور کامل، به ضرر عراق به اتمام رسید و ناوچه‌های اوزای عراقی (Osa)، در عملیات غرورآفرین مروارید که به تاریخ 7 آذر 1359 و به فرماندهی زنده‌یاد ناخدا افضلی انجام شد، تک‌تک ناوچه‌های عراقی را با موشکهای ماوریک هدف قرار داده و غرق نمودند. اگرچه دشمنی غرب سبب شد در مورد رشادتهای نیروهای هوایی و هوانیروز ایران در اوائل جنگ سخنان چندانی در محافل تخصصی غرب گفته نشود، اما درج برخی مقالات در رسانه‌های خارجی قابل ملاحظه بود تا آنجا که غربی‌ها شکست‌های سنگین عراقی‌ها را به حساب برتری تکنولوژی جنگ‌افزارهای خود نسبت به مدل روسی (که مورد استفاده عراق بود) می‌نوشتند. اما ارتش عراق نتوانست مشابه این وضعیت را در سالهای بعد جنگ برای نیروهای زمینی و دریایی ایران ایجاد کند. هرچه به سالهای انتهایی جنگ نزدیک می‌شدیم نیروی هوایی عراق بزرگتر و قدرتمندتر می‌شد اما نیروی هوایی ایران تحت تأثیر تحریم‌ها و فرسایش وحشتناک در اثر تقابل با نیروی هوایی عراق، کوچکتر و ضعیف‌تر می‌شد. در سالهای پایانی جنگ (از ۱۳۶۵ به بعد) نیروی هوایی عراق از ۶۰۰ هواپیمای جنگی مدرن (در آن زمان) بهره‌مند بود. (نیروی هوایی عراق ۳ بار به طور کامل منهدم و سپس بازسازی شد) حال آنکه ایران تنها ۱۰ درصد این تعداد را به شکل هواپیمای عملیاتی داشت، اما عراق نتوانست از نیروی هوایی خود علیه سربازان ایرانی استفادهء قابل توجهی بکند و عمدتن اقدام به بمباران و حمله به مناطق اقتصادی و مسکونی شهرهای ایران می‌نمود. ناکامی‌های نیروهای هوایی و زرهی عراق در بازپس‌گیری شهر فاو با وجود انجام صدها سورتی پرواز در روز، نشان از همین امر داشت. حتی عقب‌نشینی‌های ماههای آخر جنگ ایران در اثر برتری هوایی عراق نبود بلکه به افزایش توان آتش عراق، بزرگ شدن بیش از اندازه نیروی زمینی، استفادهء گسترده از سلاح‌های شیمیایی و پشتیبانی اطلاعاتی آمریکا از آن کشور باز می‌گشت. جنگ ایران و عراق نشان داد نیروی هوایی آموزش دیده و باتجربه و انگیزه قادر به کند کردن سرعت واحدهای زرهی و پیاده به میزان زیاد است. اما برعکس نیروی هوایی بدون ابتکار وآموزش کافی نمی‌تواند مانع پیشروی نیروهای با انگیزهء زمینی شود.

Ref: http://portal.pezdo.ir/?NID=93

geNorm

2008-03-20T15:07:00.000-04:00

[introduction

geNorm is a collection of VBA macros for Microsoft Excel to determine the most stable reference (housekeeping) genes from a set of tested candidate reference genes in a given sample panel. From this, a gene expression normalization factor can be calculated for each sample based on the geometric mean of a user-defined number of reference genes.The underlying principles and formulas are described in Vandesompele et al., Genome Biology, 2002, 'Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes'.

The full article can be read at http://genomebiology.com/2002/3/7/research/0034/[n° 5 in ranking of all time most-viewed articles published by BioMed Central][download] [5901 geNorm downloads in 94 countries]geNorm is freely available for non-commercial, academic research to be conducted at a non-profit institution. Rights to use the software outside the license agreement (e.g. commercial use) can be obtained through PrimerDesign Ltd.
the geNorm VBA applet for Microsoft Excel (Windows version, v3.5)
a beta version of geNorm for Mac (thanks to Bastian Peter, Switzerland)
geNorm user manual an Excel file containing example calculations (normalization and error propagation)[geNorm detection kits]geNorm based detection kits are available commercially from PrimerDesign Ltd. Currently available are primer sets to detect a wide variety of Homo, Mus, Rattus, Caenorhabditis, Xenopus, Arabidopsis and Ovis normalising genes.For commercial entities, kits purchased from PrimerDesign Ltd. come with a limited licence to use the geNorm software in conjunction with the kit. [geNorm citations]The following 871 papers have cited the geNorm method.[discussion]An accompanying discussion group to foster discussions between geNorm users so that they can share experiences and solutions doing gene expression normalization can be found at http://groups.yahoo.com/group/genorm[feedback]"We have been using geNorm when examining cell wall synthesis enzymes in barley tissues and are happy with the results. Thanks for generating such a useful tool and making it universally available."Neil Shirley, University of Adelaide, Australia"I am extremely impressed with this approach to normalization."Chris Tse, Sagres Discovery, USA"I can no longer analyze my PCR runs without using geNorm!"Marie-Jeanne Pillaire, CNRS, France[reference genes]Frequently used reference genes are listed here, with links to their RTPrimerDB records (containing detailed info on primer sequences, published assay, etc.) in the real-time PCR primer and probe database (see [extra]).If you want your favorite reference gene also listed here, please submit your primer/probe sequences to RTPrimerDB and notify me by email.humanACTB, B2M, GAPD, HMBS, HPRT1, RPL13A, RPL32, RPS18, SDHA, TBP, UBC, YWHAZ mouseActb, Actg1, B2m, Gapd, Hprt1, Rn18s, Tbp, Ubc ratActb, Actg2, Alb, Gapd, Hmbs, Hprt, Ubc, Ywhaz cowGapd dogGapdh zebrafishbactin1 piggapd [RTPrimerDB]You are kindly invited to submit your validated primer (and probe) sequences to the real-time PCR primer and probe database (RTPrimerDB), so that other users can benefit from your expertise.The database is available at http://medgen.ugent.be/rtprimerdb/, and is described in Pattyn et al., RTPrimerDB: the Real-Time PCR primer and probe database. Nucleic Acids Research, 2003, 31(1): 122-123 (full text)[qBase]Based on the formulas outlined on this web site (manual and example calculation file), we have developped qBase software for management and automated analysis of real-time quantitative PCR.

جمعیت ایرانیان خارج از کشور بر اساس محل اقامت

2008-03-19T22:12:00.004-04:00

مرجع: ویکی پدیا http://en.wikipedia.org/wiki/Persian_peoples

دیدار دوستان

2008-02-21T00:57:00.000-05:00

نامهایی زیبا برای کوچه و خیابانی در تهران. توانیر

If you miss Tehran

2008-02-21T00:55:00.000-05:00

معجزه ماهانه!

2008-02-19T21:17:00.002-05:00

در بسیاری از فرهنگ ها قاعدگی مظهر ناپاکی و شرم تلقی می شده و زنان در این دوره گاها از آشپزی باز داشته شده و یا از خانه بیرون رانده می شدند! با چنین زمینه تاریخی برای بسیاری عجیب بود که دریابند از خون قاعدگی می توان سلولهای پایه ای را برای نجات جان انسان ها بدست آورد. سالهاست که سلولهای پایه ای را از خون بند ناف جدا سازی و با هدف ترمیم و بازسازی بافت های بدن نگهداری می کنند. شرکت کرایو سل اینترنشنال سالهاست که تجربه جداسازی و نگهداری سلولها از خون بند ناف را دارد و حالا با هزینه ۴۹۹ دلار برای اولین بار و با هزینه ماهیانه ۹۹ دلار این امکان را فراهم کرده که خون قاعدگی را گرداوری کرده و سلولهای پایه ای را استخراج و ذخیره کنند. این شرکت اکنون در پی تاسیس بانک خون قاعدگی است. سلولهای پایه ای (بنیادی) از خون بند ناف و مغز استخوان برای بیش از یک دهه در درمان سرطان خون و بیماری های قلبی عروقی استفاده شده اند. در استفاده از سلولهای بدست آمده از خون قاعدگی توانایی این سلولها در درمان جای تحقیق و سوال بسیاری وجود دارد ولی به هر حال باید خودمان را آماده کنیم دوره پریود آغاز شده است!

منبع خبر

مقایسه چند وبسایت فارسی زبان

2008-02-17T00:42:00.005-05:00

فارس نیوز بالاترین درصد بیننده روزانه را دارد گرچه در سال ۲۰۰۸ کمی کاهش نشان می دهد. تعداد مراجعین روزانه تابناک نسبت به قبل از توقیف (بازتاب) دوبرابر شده! روزآنلاین در سال ۲۰۰۶ با افت شدیدی مواجه بوده و اکنون بیننده روزانه اش کمی کمتر از بالاترین است. بالاترین در سال ۲۰۰۷ رشد سریعی داشته و با آغاز سال جدید تعداد مراجعین کاهش داشته. بی بی سی در سال ۲۰۰۷ نسبت به قبل بیننده کمتری داشته و این کاهش در سال ۲۰۰۸ بیشتر از پیش ادامه دارد.

آه ای ديار دور ای سرزمين كودكی من

2008-01-23T20:14:00.000-05:00

آه اي ديار دور اي سرزمين كودكی من
خورشيد سرد مغرب بر من حرام باد تا آفتاب توست سرآغاز باورم
ای خاك يادگار

ای لوح جاودانه ايام
ای پاك اي زلالتر از آب و آئينه
من نقش خویش را همه جا در تو ديده ام
تا چشم بر تو دارم در خويش ننگرم
اي كاخ زرنگار اي بام لاجوردی تاريخ
فانوس ياد توست كه در خوابهاي من زير رواق غربت هميشه روشن است
برق خيال توست كه گاه گريستن دربامداد ابری من پرتو افكن است
اينجا هميشه روشني توست رهبرم
اي زادگاه مهر اي جلوگاه آتش زردشت
شب گرچه در مقابل من ايستاده است
چشمانم از بلندي طالع به سوي توست
وزپشت قله هاي مه آلوده زمين در آسمان صبح تو پيداست اخترم
اي ملك بي غروب اي مرز و بوم پير جوانبختي
ای آشیانه کهنه سیمرغ
یک روز ناگهان چون چشم من ز پنجره افتد بر آسمان
میبینم آفتاب تو را در برابرم

عشایر

2008-01-20T00:36:00.000-05:00

آموزش كمكهاي اوليه

2008-01-11T12:45:00.000-05:00

فيلم آموزشي كمكهاي اوليه و امداد به مصدومين از قبيل تنفس مصنوعي ، احياي قلبي ، باز كردن مجراي تنفسي در مواقعي كه جسمي باعث قطع شدن تنفس ميشود و رسيدگي به بيماران كه دچار سكته قلبي شده اند

لينكهاي دانلود حجم ۳۲۲ مگ

اماراتی ها و دیپلم مامایی از گوگل

2007-12-20T15:19:00.006-05:00

حتما می دونید که رستم خیلی آدم قوی و با صلابتی بوده ودر واقع اسطوره داستانهای مختلف ایرانی است/

نقل می کنند که وقتی قابله می خواسته رستم رو از مادرش جدا کنه و بیرون بیاره زورش نمی رسه و بجاش رستم دست قابله رو می گیره و می کشه داخل.

حالا اینم مثال زیاده خواهی اماراتی هاست. که دارن به دیپلم جعلی مامایی که از گوگل و نشنال جیوگرافی خریدن پز میدن!

ECG LEARNING CENTER IN CYBERSPACE

2007-12-20T13:35:00.000-05:00

ECG LEARNING CENTER IN CYBERSPACE

ATLAS OF DISEASES OF KIDNEY

2007-12-20T13:25:00.000-05:00

ATLAS OF DISEASES OF KIDNEY

Clinical Examination Videos for USMLE CS

2007-12-20T13:24:00.001-05:00

Friends here are videos required for clinical examination exam of USMLE STEP 2 CS

Download Link
http://www.gigasize.com/get.php?d=l5n19y9p2kb
http://www.gigasize.com/get.php?d=r3ns5xshygb
http://www.gigasize.com/get.php?d=lz67dsb9xsb
http://www.gigasize.com/get.php?d=bv8sdrd7woc

All files are in rar format and are password protected

Password:
sajjad

These videos will be of help for any clinical examination exam, and for internship and clinical rotations.

Obstetric Examination Video

2007-12-20T13:08:00.000-05:00

Obstetric Examination

CPR Video

2007-12-20T13:06:00.000-05:00

CPR Video

Physical Examination (Video Clips)

2007-12-20T12:56:00.000-05:00

Chest Examination:

Introduction
http://www.smso.net/media/02V/02V01.WMV

Assessment of the Chest, Respirations, and the Posterior Thorax
http://www.smso.net/media/02V/02V02.WMV

Assessment of the Posterior Thorax
http://www.smso.net/media/02V/02V03.WMV

Percussion of the Posterior Thorax
http://www.smso.net/media/02V/02V04.WMV

Review of Breath Sounds
http://www.smso.net/media/02V/02V05.WMV

Adventitious Breath Sounds
http://www.smso.net/media/02V/02V06.WMV

Auscultation of the Posterior Thorax
http://www.smso.net/media/02V/02V07.WMV

Assessment of the Anterior Thorax
http://www.smso.net/media/02V/02V08.WMV

Percussion of the Anterior Thorax
http://www.smso.net/media/02V/02V09.WMV

Auscultation of the Anterior Thorax
http://www.smso.net/media/02V/02V10.WMV

Summary
http://www.smso.net/media/02V/02V11.WMV
__________________
Cardiovascular: Neck Vessels and Heart:

Introduction
http://www.smso.net/media/01V/01V01.WMV

Examination of the Neck Vessels
http://www.smso.net/media/01V/01V02.WMV

Examination of the Heart
http://www.smso.net/media/01V/01V03.WMV

Review of Heart Sounds
http://www.smso.net/media/01V/01V04.WMV

Auscultation of the Heart
http://www.smso.net/media/01V/01V05.WMV

Heart Sounds: S1, S2
http://www.smso.net/media/01V/01V06.WMV

Heart Sounds: S3, S4, murmurs
http://www.smso.net/media/01V/01V07.WMV

Summary
http://www.smso.net/media/01V/01V08.WMV

Cardiovascular: Peripheral Vascular System:

Introduction
http://www.smso.net/media/05V/05V01.WMV

Examination of the Arms
http://www.smso.net/media/05V/05V02.WMV

Examination of the Legs
http://www.smso.net/media/05V/05V03.WMV

Examination of the Legs (continued)
http://www.smso.net/media/05V/05V04.WMV

Summary
http://www.smso.net/media/05V/05V05.WMV
__________________
Abdomen Examination:
Introduction
http://www.smso.net/media/04V/04V01.WMV

Inspection of the Abdomen
http://www.smso.net/media/04V/04V02.WMV

Auscultation of the Abdomen
http://www.smso.net/media/04V/04V03.WMV

Percussion of the Abdomen
http://www.smso.net/media/04V/04V04.WMV

Palpation of the Abdomen
http://www.smso.net/media/04V/04V05.WMV

Examination of the Liver
http://www.smso.net/media/04V/04V06.WMV

Examination of the Spleen
http://www.smso.net/media/04V/04V07.WMV

Examination of the Kidneys and Aorta
http://www.smso.net/media/04V/04V08.WMV

Summary
http://www.smso.net/media/04V/04V09.WMV
__________________
Neurological: Cranial Nerves and Sensory System:

Introduction
http://www.smso.net/media/06V/06V01.WMV

General Observation of Neurological Status
http://www.smso.net/media/06V/06V02.WMV

Cranial Nerves I and II
http://www.smso.net/media/06V/06V03.WMV

Cranial Nerves III, IV, and VI
http://www.smso.net/media/06V/06V04.WMV

Cranial Nerves V and VII
http://www.smso.net/media/06V/06V05.WMV

Cranial Nerve VII
http://www.smso.net/media/06V/06V06.WMV

Cranial Nerves IX, X, XI, and XII
http://www.smso.net/media/06V/06V07.WMV

Sensory Assessment: Pain, Temperature, and Light Touch Sensations
http://www.smso.net/media/06V/06V08.WMV

Sensory Assessment: Vibration Sensation and Position Sense
http://www.smso.net/media/06V/06V09.WMV

Sensory Assessment: Discriminatory Sensations
http://www.smso.net/media/06V/06V10.WMV

Summary
http://www.smso.net/media/06V/06V11.WMV
__________________
Neurologic: Motor System and Reflexes:

Introduction
http://www.smso.net/media/03V/03V01.WMV

Assessment of the Motor System: Upper Extremities
http://www.smso.net/media/03V/03V02.WMV

Assessment of the Motor System: Lower Extremities
http://www.smso.net/media/03V/03V03.WMV

Assessment of Coordination
http://www.smso.net/media/03V/03V04.WMV

Romberg Test; Testing for Pronator Drift
http://www.smso.net/media/03V/03V05.WMV

Assessment of Reflexes
http://www.smso.net/media/03V/03V06.WMV

Supine Assessment of Reflexes
http://www.smso.net/media/03V/03V07.WMV

Further Testing of Reflexes
http://www.smso.net/media/03V/03V08.WMV

Summary
http://www.smso.net/media/03V/03V09.WMV
__________________
Muscoskeletal System:

Introduction
http://www.smso.net/media/09V/09V01.WMV

Assessment of the Head and Neck
http://www.smso.net/media/09V/09V02.WMV

Assessment of the Hands and Wrists
http://www.smso.net/media/09V/09V03.WMV

Assessment of the Elbows
http://www.smso.net/media/09V/09V04.WMV

Assessment of the Shoulders and Related Structures
http://www.smso.net/media/09V/09V05.WMV

Assessment of the Feet and Ankles
http://www.smso.net/media/09V/09V06.WMV

Assessment of the Legs
http://www.smso.net/media/09V/09V07.WMV

Assessment of the Hips
http://www.smso.net/media/09V/09V08.WMV

Assessment of the Spine
http://www.smso.net/media/09V/09V09.WMV

Summary
http://www.smso.net/media/09V/09V10.WMV
__________________
Head, Eyes and Ears:

Introduction
http://www.smso.net/media/07V/07V01.WMV

Inspection of the Head
http://www.smso.net/media/07V/07V02.WMV

Examination of the Eyes: Visual Acuity and Visual Fields
http://www.smso.net/media/07V/07V03.WMV

Inspection of the Eyes
http://www.smso.net/media/07V/07V04.WMV

Assessment of the Extraocular Muscles
http://www.smso.net/media/07V/07V05.WMV

Ophthalmoscopic Examination
http://www.smso.net/media/07V/07V06.WMV

Examination of the Ears
http://www.smso.net/media/07V/07V07.WMV

Assessment of Hearing
http://www.smso.net/media/07V/07V08.WMV

Summary
http://www.smso.net/media/07V/07V09.WMV
__________________
Nose, Mouth and Neck:

Introduction
http://www.smso.net/media/08V/08V01.WMV

Inspection of the Nose
http://www.smso.net/media/08V/08V02.WMV

Inspection of the Mouth
http://www.smso.net/media/08V/08V03.WMV

Inspection of the Neck
http://www.smso.net/media/08V/08V04.WMV

Summary
http://www.smso.net/media/08V/08V05.WMV
__________________

Online surgery videos

2007-12-19T11:25:00.000-05:00

I bet many of medical students, are curious about surgical procedures and wish they can see it live instead of just reading theoritically about it.

Anyways, your wishes are about to come true...these are some important video archives libraries with a heavy load of surgical procedures live videos...

Enjoy...

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Lifespan - Online surgery videos

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MedlinePlus:Videos of Surgical Procedures

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Cardiothoracic Surgery: University of southern California keck school of medicine

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The Heart Surgery Forum

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Spine Universe: spinal surgery

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Understandspinesurgery.com: spinal surgery procedures

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Laparoscopy.com

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Hand Surgery Videos

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Medicdirect: downloadable surgical operations videos

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The Royal College Of Surgeons Of Edinburgh:

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Somerset: Medical center - Surgical procedure videos - This site contains only surgical op simulation videos [no real operations]

-----------------------------------------

Making of a PCR Internal Standard

2007-12-18T15:31:00.000-05:00

A. Basics

It is possible to make RT-PCR quantitative, so do not listen to non-PCR'ers. However, you need to find a way to negate the tube-to-tube variability that is inherent in the amplification process. The only good way to do this is to add an internal standard (IS) each tube. There are many ways to make internal standards; the method shown below has several advantages in that it is adaptable to any primer sequences and is easy to perform. How you make an internal standard is not as important as its properties. First it should be amplified with the same efficiency as the cDNA being quantified. This is generally done by having the IS and target with the same primer recognition sequences and by making the two PCR products of similar length. Also, you need to resolve the IS PCR product from the target PCR product. This may be done by changing a restriction enzyme site in the IS or by making the IS of a slightly different length. Another factor when using an IS for RT-PCR is to start with a RNA IS as well to negate variability in the cDNA synthesis. The method used in our lab does fulfill these requirements, as you will see.

B. Design of Internal Standard Primers

1. For basics of internal standard synthesis see Figure 1 of Vanden Heuvel et al. (Biotechniques). The design of the internal standard is: Forward Primer--T7 promoter, target forward primer, spacer forward primer; Reverse Primer--spacer reverse primer, target reverse primer, poly(T)15.

NOTE #1: The spacer primer sequences are designed so that ANY sequence can be inserted. The key is to find a primer pair (the spacer primers) that will ultimately result in a PCR product that is different from that of the target. In the Biotechniques paper we used human GSTMu as our linker primers. We have also used interferon-g and b-globin sequences to make primers. The key is to find primers that work and they give the appropriate size product. Generally, we have browsed through commercial catalogs to find sequences.
NOTE #2: The primers used in the making of the IS are quite long (around 60 bp) but only the 3' end (that containing the linker or spacer primers) will anneal and amplify. The rest of the primer will be incorporated into the PCR product.

2. It is possible to design internal standards with more than one gene per internal standard. Basically you can have two genes per internal standard primer and you can do two rounds of application. For example, round one forward primer Target 2 FP, Target 1 FP and Spacer FP; round one reverse primer Spacer RP, Target 1 RP and Target 2 RP. Second round forward primer, T7, Target 3 FP, Target 2 FP. Second round reverse primer, Target 2 RP, Target 3 RP and PolyT. Much more thought goes into designing these primers.

3. We use human genomic DNA as a source of template for making internal standard to use for rat genes. It probably does not matter, but it makes us feel safer.

C. Internal Standard Amplification

1. Amplify human genomic DNA (10 ng, if this is the source of your linker primers) using the reverse and forward internal standard (IS) primers (See appendix IV). Do multiple PCR reactions for each IS. A touch-down and 35 cycles is suggested.

2. Pool the multiple IS reactions and purify using Magic PCR Prep Purification system (Promega) or Microcon 100 spin columns following the manufacturers suggestions. This step will remove unused primers. Analyze on an agarose gel. At this point you may not be able to see a PCR band, depending on the efficiency of the primers.

3. Dilute PCR products 1:100 and amplify multiple tubes (5-6) as shown in step 1.

4. Pool and purify PCR products as stated in Step 2. Analyze PCR products on an agarose gel. If the product is not clean enough, gel purify the appropriate band. If a nice clean IS band is observed, continue with the in vitro transcription protocol as listed below.

D. In vitro transcription

1. Prepare the following mix (from Promega's Gemini II kit)
Transcription buffer 20 ul
100 mM DTT 10 ul
rRNasin 2.5 ul
rATP/rCTP/rGTP/rUTP 5 ul each
IS PCR product 45 ul
T7 RNA polymerase 2 ul

Incubate for 1-2 hr at 37 C

2. Add 2 ul RQ1 RNase-Free Dnase. Incubate for 15-30 min at 37 C.

3. Add 100 ul TE-buffered phenol. Vortex for 1 min and centrifuge at 12,000 g for 10 min.

4. Transfer upper phase to a fresh tube. Add 100 ul chloroform/isoamyl alcohol (24:1).

5. Transfer upper phase to a fresh tube. Add 50 ul 10 N ammonium acetate (pH 4.0) and 500 ul ethanol. Precipitate at -20 C for 30 min.

6. Spin at 12,000 g for 10 min. Wash with 70% ethanol.

7. Quantitate RNA using absorbance at 260 nm. [Note: to quantitate RNA use the following formula: ABS260 x 0.04 x dilution factor = ug/ul].

8. How to calculate molecules/ul of IS:

ug/ul
____________________________________ x 6.02 E 17 mlcls/umole
(330 ug/umol/bp x bp IS)

The 330 x bp is an approximation for the molecular weight of the internal standard. For example, a 0.1 mg/ml solution of a 400 bp IS would be 4.56 x 1011 mlcls/ul.

Essentials of Real Time PCR

2007-12-18T15:30:00.000-05:00

About Real-Time PCR Assays
Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as
it occurs ( i.e., in real time). Data is therefore collected throughout the PCR process, rather than
at the end of the PCR. This completely revolutionizes the way one approaches PCR-based
quantitation of DNA and RNA. In real-time PCR, reactions are characterized by the point in time
during cycling when amplification of a target is first detected rather than the amount of target
accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic
acid target, the sooner a significant increase in fluorescence is observed. In contrast, an
endpoint assay (also called a “plate read assay”) measures the amount of accumulated PCR
product at the end of the PCR cycle.
About Sequence Detection Chemistries
Overview Applied Biosystems has developed two types of chemistries used to detect PCR
products using Sequence Detection Systems (SDS) instruments:
• TaqMan® chemistry (also known as “fluorogenic 5´ nuclease chemistry”)
• SYBR® Green I dye chemistry
TaqMan® Chemistry
The TaqMan chemistry uses a fluorogenic probe to enable the detection of a specific PCR
product as it accumulates during PCR cycles.
Assay Types that Use TaqMan Chemistry
The TaqMan chemistry can be used for the following assay types:
• Quantitation, including:
– One-step RT-PCR for RNA quantitation
– Two-step RT-PCR for RNA quantitation
– DNA/cDNA quantitation
• Allelic Discrimination
• Plus/Minus using an IPC
SYBR Green I Dye Chemistry
The SYBR Green I dye chemistry uses SYBR Green I dye, a highly specific, double-stranded
DNA binding dye, to detect PCR product as it accumulates during PCR cycles.
The most important difference between the TaqMan and SYBR Green I dye chemistries is that
the SYBR Green I dye chemistry will detect all double-stranded DNA, including non-specific
reaction products. A well-optimized reaction is therefore essential for accurate results.
Assay Types that Use SYBR Green I Dye Chemistry
The SYBR Green I dye chemistry can be used for the following assay types:
• Quantitation, including:
– One-step RT-PCR for RNA quantitation
– Two-step RT-PCR for RNA quantitation
– DNA/cDNA quantitation
Page 2 of 8
TaqMan Chemistry
Background
Initially, intercalator dyes were used to measure real-time PCR products. The primary
disadvantage to these type of probes is that they detect accumulation of both specific and nonspecific
PCR products.
Development of TaqMan Chemistry
Real-time systems for PCR were improved by the introduction of fluorogenic-labeled probes that
use the 5´ nuclease activity of Taq DNA polymerase. The availability of these fluorogenic probes
enabled the development of a real-time method for detecting only specific amplification products.
The development of fluorogenic labeled probes also made it possible to eliminate post-PCR
processing for the analysis of probe degradation.
How TaqMan Sequence Detection Chemistry Works
The TaqMan chemistry uses a fluorogenic probe to enable the detection of a specific PCR
product as it accumulates during PCR. Here’s how it works:
Step Process
1. An oligonucleotide probe is constructed containing a reporter fluorescent dye on the 5´ end and a
quencher dye on the 3´ end. While the probe is intact, the proximity of the quencher dye greatly
reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer
(FRET) through space.
2. If the target sequence is present, the probe anneals downstream from one of the primer sites and is
cleaved by the 5´ nuclease activity of Taq DNA polymerase as this primer is extended.
3. This cleavage of the probe:
• Separates the reporter dye from the quencher dye, increasing the reporter dye signal.
• Removes the probe from the target strand, allowing primer extension to continue to the
end of the template strand. Thus, inclusion of the probe does not inhibitthe overall PCR
process.
4. Additional reporter dye molecules are cleaved from their respective probes with each cycle,
resulting in an increase in fluorescence intensity proportional to the amount of amplicon produced.
Page 3 of 8
Two Types of TaqMan® Probes
Applied Biosystems offers two types of TaqMan probes:
• TaqMan® probes (with TAMRA dye as the quencher dye)
• TaqMan® MGB probes
TaqMan MGB Probes Recommended for Allelic Discrimination Assays
Applied Biosystems recommends the general use of TaqMan MGB probes for allelic
discrimination assays, especially when conventional TaqMan probes exceed 30 nucleotides. The
TaqMan MGB probes contain:
• A nonfluorescent quencher at the 3´ end - The SDS instruments can measure the
reporter dye contributions more precisely because the quencher does not fluoresce.
• A minor groove binder at the 3´ end - The minor groove binder increases the melting
temperature (Tm) of probes, allowing the use of shorter probes.
Consequently, the TaqMan MGB probes exhibit greater differences in Tm values between
matched and mismatched probes, which provides more accurate allelic discrimination.
Advantages of TaqMan Chemistry
The advantages of the TaqMan chemistry are as follows:
• Specific hybridization between probe and target is required to generate fluorescent signal
• Probes can be labeled with different, distinguishable reporter dyes, which allows
amplification of two distinct sequences in one reaction tube
• Post-PCR processing is eliminated, which reduces assay labor and material costs
Disadvantage of TaqMan Chemistry
The primary disadvantage of the TaqMan chemistry is that the synthesis of different probes is
required for different sequences.
SYBR Green I Dye Chemistry
Background
Small molecules that bind to double-stranded DNA can be divided into two classes:
• Intercalators
• Minor-groove binders
Regardless of the binding method, there are two requirements for a DNA binding dye
for real-time detection of PCR:
• Increased fluorescence when bound to double-stranded DNA
• No inhibition of PCR
Applied Biosystems has developed conditions that permit the use of the SYBR Green I dye in
PCR without PCR inhibition and increased sensitivity of detection compared to ethidium bromide.
Page 4 of 8
How the SYBR Green I Dye Chemistry Works
The SYBR Green I dye chemistry uses the SYBR Green I dye to detect polymerase chain
reaction (PCR) products by binding to double-stranded DNA formed during PCR. Here’s how it
works:
Step Process
1. When SYBR Green I dye is added to a sample, it immediately binds to all double-stranded DNA
present in the sample.
2. During the PCR, AmpliTaq Gold® DNA Polymerase amplifies the target sequence, which creates
the PCR products, or “amplicons.”
3. The SYBR Green I dye then binds to each new copy of double-stranded DNA.
4. As the PCR progresses, more amplicons are created. Since the SYBR Green I dye binds to all
double-stranded DNA, the result is an increase in fluorescence intensity proportionate to the
amount of PCR product produced.
Advantages of SYBR Green I Dye
The advantages of the SYBR Green I dye chemistry are as follows:
• It can be used to monitor the amplification of any double-stranded DNA sequence.
• No probe is required, which reduces assay setup and running costs.
Disadvantage of SYBR Green I Dye
The primary disadvantage of the SYBR Green I dye chemistry is that it may generate false
positive signals; i.e., because the SYBR Green I dye binds to any double-stranded DNA, it can
also bind to nonspecific double-stranded DNA sequences.
Additional Consideration
Another aspect of using DNA binding dyes is that multiple dyes bind to a single amplified
molecule. This increases the sensitivity for detecting amplification products. A consequence of
multiple dye binding is that the amount of signal is dependent on the mass of double-stranded
DNA produced in the reaction. Thus, if the amplification efficiencies are the same, amplification of
a longer product will generate more signal than a shorter one. This is in contrast to the use of a
fluorogenic probe, in which a single fluorophore is released from quenching for each amplified
molecule synthesized, regardless of its length.
About Quantitation Assays
What Is a Quantitation Assay?
A Quantitation Assay is a real-time PCR assay. It measures (quantitates) the amount of a nucleic
acid target during each amplification cycle of the PCR. The target may be DNA, cDNA, or RNA.
There are three types of Quantitation Assays discussed in this chemistry guide:
• DNA/cDNA quantitation
• RNA quantitation using one-step reverse transcription polymerase chain reaction (RTPCR)
• RNA quantitation using two-step RT-PCR
Terms Used in Quantitation Analysis
Amplicon A short segment of DNA generated by the PCR process
Amplification plot The plot of fluorescence signal versus cycle number
Baseline The initial cycles of PCR, in which there is little change in fluorescence signal
CT (threshold cycle) The fractional cycle number at which the fluorescence passes the fixed threshold
Page 5 of 8
NTC (no template control) - A sample that does not contain template. It is used to verify amplification
quality.
Nucleic acid target (also called “target template”) - DNA or RNA sequence that you wish to
amplify
Passive reference A dye that provides an internal reference to which the reporter dye signal can be
normalized during data analysis. Normalization is necessary to correct for forestallment
fluctuations caused by changes in concentration or volume. A passive reference dye is included in all SDS
PCR reagent kits.
Rn (normalized reporter) The fluorescence emission intensity of the reporter dye divided by the
fluorescence emission intensity of the passive reference dye
Rn+ The Rn value of a reaction containing all components, including the template
Rn- The Rn value of an un-reacted sample. The Rn- value can be obtained from:
• The early cycles of a real-time PCR run (those cycles prior to a detectable increase in
fluorescence), OR
• A reaction that does not contain any template
ΔRn (delta Rn) The magnitude of the signal generated by the given set of PCR conditions. The ΔRn value is
determined by the following formula:
(Rn+) – (Rn-)
Standard A sample of known concentration used to construct a standard curve. By running standards of
varying concentrations, you create a standard curve from which you can extrapolate the quantity of an
unknown sample.
Threshold The average standard deviation of Rn for the early PCR cycles, multiplied by an adjustable factor.
The threshold should be set in the region associated with an exponential growth of PCR product.
Unknown A sample containing an unknown quantity of template. This is the sample whose quantity you
want to determine.
How Real-Time PCR Quantitation Assays Work
In the initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline
for the amplification plot. An increase in fluorescence above the baseline indicates the detection
of accumulated target. A fixed fluorescence threshold can be set above the baseline. The
parameter CT (threshold cycle) is defined as the fractional cycle number at which the fluorescence
passes the fixed threshold.
Absolute vs. Relative Quantitation
Overview
When calculating the results of your quantitation assays, you can use either absolute or relative
quantitation.
What is Absolute Quantitation?
The absolute quantitation assay is used to quantitate unknown samples by interpolating their
quantity from a standard curve.
Example
Absolute quantitation might be used to correlate viral copy number with a disease state. It is of
interest to the researcher to know the exact copy number of the target RNA in a given biological
sample in order to monitor the progress of the disease.
Absolute quantitation can be performed with data from all of the SDS instruments, however, the
absolute quantities of the standards must first be known by some independent means.
Page 6 of 8
What is Relative Quantitation?
A relative quantitation assay is used to analyze changes in gene expression in a given sample
relative to another reference sample (such as an untreated control sample).
Example
Relative quantitation might be used to measure gene expression in response to a chemical
(drug). The level of gene expression of a particular gene of interest in a chemically treated
sample would be compared relative to the level of gene expression an untreated sample.
Calculation Methods for Relative Quantitation
Relative quantitation can be performed with data from all of the SDS instruments. The calculation
methods used for relative quantitation are:
• Standard curve method
• Comparative CT method
Determining Which Method to Use
All methods can give equivalent results. When determining which method you want to use, note
the following:
• Running the target and endogenous control amplifications in separate tubes and using
the standard curve method of analysis requires the least amount of optimization and
validation.
• To use the comparative CT method, a validation experiment must be run to show that the
efficiencies of the target and endogenous control amplifications are approximately equal.
The advantage of using the comparative CT method is that the need for a standard curve
is eliminated. This increases throughput because wells no longer need to be used for the
standard curve samples. It also eliminates the adverse effect of any dilution errors made
in creating the standard curve samples.
• To amplify the target and endogenous control in the same tube, limiting primer
concentrations must be identified and shown not to affect CT values. By running the two
reactions in the same tube, throughput is increased and the effects of pipetting errors are
reduced.
Terms Used
The following terms are used in this discussion of absolute and relative quantitation:
Standard A sample of known concentration used to construct a standard curve.
Reference A passive or active signal used to normalize experimental results. Endogenous and exogenous
controls are examples of active references. Active reference means the signal is generated as the result of
PCR amplification. The active reference has its own set of primers and probe.
Endogenous control – This is an RNA or DNA that is present in each experimental sample as isolated. By
using an endogenous control as an active reference, you can normalize quantitation of a messenger RNA
(mRNA) target for differences in the amount of total RNA added to each reaction.
Exogenous control – This is a characterized RNA or DNA spiked into each sample at a known
concentration. An exogenous active reference is usually an in vitro construct that can be used as an internal
positive control (IPC) to distinguish true target negatives from PCR inhibition. An exogenous reference can
also be used to normalize for differences in efficiency of sample extraction or complementary DNA (cDNA)
synthesis by reverse transcriptase. Whether or not an active reference is used, it is important to use a
passive reference containing the dye ROX in order to normalize for non-PCR-related fluctuations in
fluorescence signal.
Normalized amount of target
A unitless number that can be used to compare the relative amount of target in different samples.
Calibrator A sample used as the basis for comparative results.
Page 7 of 8
Standard Curve Method for Relative Quantitation
Overview
It is easy to prepare standard curves for relative quantitation because quantity is expressed
relative to some basis sample, such as the calibrator. For all experimental samples, target
quantity is determined from the standard curve and divided by the target quantity of the calibrator.
Thus, the calibrator becomes the 1× sample, and all other quantities are expressed as an n-fold
difference relative to the calibrator. As an example, in a study of drug effects on expression, the
untreated control would be an appropriate calibrator.
Critical Guidelines
The guidelines below are critical for proper use of the standard curve method for relative
quantitation:
• It is important that stock RNA or DNA be accurately diluted, but the units used to express
this dilution are irrelevant. If two-fold dilutions of a total RNA preparation from a control
cell line are used to construct a standard curve, the units could be the dilution values 1,
0.5, 0.25, 0.125, and so on. By using the same stock RNA or DNA to prepare standard
curves for multiple plates, the relative quantities determined can be compared across the
plates.
• It is possible to use a DNA standard curve for relative quantitation of RNA. Doing this
requires the assumption that the reverse transcription efficiency of the target is the same
in all samples, but the exact value of this efficiency need not be known.
• For quantitation normalized to an endogenous control, standard curves are prepared for
both the target and the endogenous reference. For each experimental sample, the
amount of target and endogenous reference is determined from the appropriate standard
curve. Then, the target amount is divided by the endogenous reference amount to obtain
a normalized target value. Again, one of the experimental samples is the calibrator, or
1× sample. Each of the normalized target values is divided by the calibrator normalized
target value to generate the relative expression levels.
Endogenous Control
Amplification of an endogenous control may be performed to standardize the amount of sample
RNA or DNA added to a reaction. For the quantitation of gene expression, researchers have used
ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal RNA (rRNA), or other
RNAs as an endogenous control.
Standards
Because the sample quantity is divided by the calibrator quantity, the unit from the standard curve
drops out. Thus, all that is required of the standards is that their relative dilutions be known. For
relative quantitation, this means any stock RNA or DNA containing the appropriate target can be
used to prepare standards.
Comparative CT method for Relative Quantitation
The comparative CT method is simlar to that standard curve method, except it uses the arithmetic
formula, 2-􀁕􀁕C
T to achieve the same result for relative quantitation.
Arithmetic Formulas:
For the comparative CT method to be valid, the efficiency of the target amplification (your gene of
interest) and the efficiency of the reference amplification (your endogenous control) must be
approximately equal.
Page 8 of 8
For more information on using the comparative CT method for relative quantitation, please refer to
User Bulletin #2: Relative Quantitation of Gene Expression (PN 4303859).
Standard Curve Method for Absolute Quantitation
Overview
The standard curve method for absolute quantitation is similar to the standard curve method for
relative quantitation, except the absolute quantities of the standards must first be known by some
independent means.
Critical Guidelines
The guidelines below are critical for proper use of the standard curve method for absolute
quantitation:
• It is important that the DNA or RNA be a single, pure species. For example, plasmid DNA
prepared from E. coli often is contaminated with RNA, which increases the A260
measurement and inflates the copy number determined for the plasmid.
• Accurate pipetting is required because the standards must be diluted over several orders
of magnitude. Plasmid DNA or in vitro transcribed RNA must be concentrated in order to
measure an accurate A260 value. This concentrated DNA or RNA must then be diluted
106–1012 -fold to be at a concentration similar to the target in biological samples.
• The stability of the diluted standards must be considered, especially for RNA. Divide
diluted standards into small aliquots, store at –80 °C, and thaw only once before use.
• It is generally not possible to use DNA as a standard for absolute quantitation of RNA
because there is no control for the efficiency of the reverse transcription step.
Standards
The absolute quantities of the standards must first be known by some independent
means. Plasmid DNA and in vitro transcribed RNA are commonly used to prepare absolute
standards. Concentration is measured by A260 and converted to the number of copies using the
molecular weight of the DNA or RNA.

REAGENTS AND METHODS FOR ISOLATION OF PURIFIED RNA

2007-12-18T14:55:00.000-05:00

REAGENTS AND METHODS FOR ISOLATION OF PURIFIED RNA FIELD OF THE INVENTION The invention is directed to compositions and methods that enhance isolation of purified RNA from biological samples. BACKGROUND Isolation of pure, intact RNA is a critical step for analysis of gene expression in molecular biology, clinical, and biotechnology applications. Methods of RNA isolation have been developed in an attempt to achieve this goal. The most frequently used methods for RNA isolation are based on phenol extraction, precipitation from chaotropic salt solutions, and adsorption on silica (Ausubel et al, 2002), reviewed in my U.S. Patent Nos. 4,843,155; 5,346,994; and 5,945,515. The method described in the '155 patent is frequently referred to as the single- step method and extracts RNA with a phenol-guanidine solution at pH 4. Its effectiveness and simplicity make the single-step method the most frequently used method for isolating RNA. An improvement of the single-step method, described in my subsequent '994 patent, allowed simultaneous isolation of RNA, DNA, and proteins from the same sample by phenol-guanidine extraction at pH 4 - 6. A biological sample is homogenized and the homogenate is subjected to phase separation using a hydrophobic organic solvent such as chloroform or bromochloropropane. Following centrifugation, the mixture separates into the top aqueous phase containing RNA, and the interphase and organic phase containing DNA and proteins. The aqueous phase is collected and RNA is precipitated and washed with alcohol. In the single-step method described in the '155 and '994 patents, a careful collection of the separated aqueous phase is critical for the quality of the isolated RNA. Small amounts of the interphase and organic phase can be easily removed together with the aqueous phase, which results in contamination of the isolated RNA with DNA and proteins. Also, collection of the aqueous phase requires a manual approach, which is an obstacle in adapting the single- step method for automation. The reagents and methods described in the '155 and '994 patents provide substantially pure, undegraded RNA. However.-RNA isolated according to the '155 and '994 patents contains a residual amount of genomic DNA, which can be detected by reverse transcription-polymerase chain reaction assay (RT-PCR). Thus, RNA isolated in accord with the '155 and '994 patents must be further purified to render it DNA-free (Guan at al, 2003; Girotti and Zingg, 2003). The contaminating genomic DNA serves as a matrix for DNA polymerase, yielding additional amplification products and distorting RNA-dependent RT-PCR. The DNA contamination in RT-PCR can be only partially alleviated by using a set of primers encompassing exon-intron sequences in the genomic DNA because the presence of pseudogenes, containing no introns, makes this approach unreliable (Mutimer 1998). Modifications to the single-step method have improved the quality of the isolated RNA. In one modification, RT-PCR inhibitors were removed by adding a lithium chloride precipitation step (Puissant, 1990; Mathy, 1996). In another modification, alcohol precipitation of RNA in the presence of salt increased purity of the isolated RNA (Chomczynski, 1995). These modifications, however, were not effective in removing DNA contamination. A common practice for removing contaminating DNA is to treat an RNA- containing sample with deoxyribonuclease (DNase). Following DNase treatment, the RNA- containing sample is extracted sequentially with phenol and chloroform. In an effort to limit DNA contamination, an additional DNA precipitation step was included in the single-step method. The contaminating DNA was precipitated from the aqueous phase by adding one-third the volume of 95%w/w ethanol (Siebert, 1993). The final concentration of ethanol was about 24% w/w. The author indicated that, at this low ethanol concentration, DNA was precipitated while RNA remained in solution. RNA was precipitated from the solution by adding additional alcohol. This protocol, however, yielded RNA that was still contaminated with DNA, evidenced as a visible band upon analyzing the isolated RNA on an agarose gel stained with ethidium bromide and by RT- PCR. In another effort to diminish DNA contamination and improve the quality of RNA in the single-step method, Monstein (1995) in a laborious procedure increased the pH of the phenol extraction to pH 4.1 - 4.7 and treated the sample with proteinase K, followed by another round of phenol extraction, precipitation, and ethanol wash. Despite this prolonged procedure, DNase treatment was still necessary to obtain DNA-free RNA ready for use in RT-PCR. Separating RNA from DNA was also achieved by phenol extraction at pH 4 without adding guanidine salts (Kedzierski, 1991). However, the absence of guanidine salts during the procedure made RNA susceptible to ribonuclease (RNase), thereby degrading the RNA. A later improvement of this protocol employed phenol extraction buffer at pH 4.2 in the presence of sodium dodecyl sulfate (Chattopadhyay et al., 1993). DNase treatment was also required in the RNA isolation method using a combination of the single-step method followed by the silica column procedure (Bonham, 1996). The use of this double purification protocol decreased DNA contamination, but the isolated RNA still contained genomic DNA that was detected by RT-PCR. Another method for isolating RNA used a monophase aqueous solution containing 10%w/w to 60%w/w phenol (U.S. Patent Application Publication 20030204077). In the absence of chaotropes, 15%w/w to 55%w/w monoalcohol, diol, or polyol was used to keep phenol in aqueous solution Thus, a residual amount of DNA present in RNA isolated by the methods described in the '155 and '994 patents made it necessary to extend the procedure by including DNase treatment. This diminished the usefulness of the methods by prolonging procedures and unnecessarily exposing RNA to the possibility of degradation during DNase treatment and additional purification steps. However, removing residual DNA from RNA preparations is needed for RT-PCR based microarray determination of gene expression. Previous methods for isolating RNA, as described in the '155 and '994 patents, were based on phenol extraction performed at pH 4 or higher. None of the previous modifications of the single-step method attempted to improve the quality of RNA by performing phenol extraction at a pH below 4. To the contrary, pH 4 as used in the first '155 patent was increased in the next '994 patent to a pH ranging from 4 to 6. Similarly, the protocol described by Monstein (1995) increased the pH of the phenol extraction to pH 4.7. Another elaborate attempt to improve the single-step method increased the pH of the guanidine-phenol extract to pH 5.2 (Suzuki, 2003). An alternative to the single-step method of RNA isolation was disclosed in U.S. Patent No. 5,973,137, using non-chaotropic acidic salts. However, the single-step phenol extraction method is still the most frequently used method for RNA isolation. A publication describing the single-step method (Chomczynski 1987) is the fourth most cited paper in the database of the American Chemical Society and Institute for Scientific Information, and the most cited paper published within the last twenty years (CAS 2003, American Chemical Society). New methods to enhance purity of isolated RNA are thus desirable. SUMMARY OF THE INVENTION The present invention discloses reagents and methods capable of isolating from a biological sample RNA that is substantially free of DNA and thus ready for reverse transcriptase polymerase chain reaction (RT-PCR). Such RNA is termed substantially pure RNA, and is required for proper diagnosis of gene expression in clinical, research and other applications. One embodiment is a phase separation method using acidic phenol, with RNA separating in the aqueous phase. This is based on the unexpected finding that substantially pure RNA can be isolated by phenol extraction performed at pH below 4. Another embodiment is acidic phenol precipitation of DNA and protein, with RNA remaining in the soluble fraction. This is based on the unexpected finding that certain concentrations of acidic phenol selectively precipitate DNA, proteins and other cellular components, leaving RNA remaining in a soluble form. The use of acidic phenol for selectively precipitating DNA and proteins eliminates the need for phase separation and also eliminates the use of toxic phase-separation solvents. This approach significantly simplifies the RNA isolation process. Another embodiment is selective RNA precipitation from solutions containing phenol, a chaotrope, and a low volume of an organic solvent. This embodiment may be used to selectively precipitate RNA molecules up to about 200 nucleotides. Shorter RNA molecules (lower molecular weight RNA) and/or DNA may also be recovered. DNA may also be recovered from the sample by increasing the concentration of organic solvent to at least about 50%w/w. Another embodiment is RNA precipitation from solutions containing at least one salt by adjusting the pH of the solution to a maximum pH of 3.3. RNA isolated by the inventive compositions and methods can be used directly for RT-PCR because it has a higher purity, that is, there is less contamination of RNA by DNA and/or protein, in comparison to previous methods for RNA isolation such as methods disclosed in U.S. Patent Nos. 4,843,155; 5,346,994; and 5,945,515, each of which is expressly incorporated herein by reference in its entirety. The RNA that is isolated may be single stranded (ssRNA) or double stranded (dsRNA), and may be isolated from a variety of biological sources, including animals, plants, yeasts, bacteria, and viruses. RNA isolated by the inventive methods and using the inventive compositions may be used in molecular biology, biotechnology, and clinical sciences. In addition, the inventive reagents may be used alone or in combination with other methods for isolating substantially pure DNA (DNA substantially free of RNA and protein), and substantially pure proteins (proteins substantially free of RNA and DNA). These and other advantages will be apparent in light of the following detailed description and examples. DETAILED DESCRIPTION Methods and compositions to prepare purified RNA from biological samples are disclosed. A biological sample is any sample from a biological source, whether in vivo, in vitro, or ex vivo. Samples may be from humans, animals, plants, bacteria, viruses, fungi, parasites, mycoplasmas, etc. Purified RNA is RNA that is substantially undegraded and free of DNA contamination when assayed by reverse transcriptase polymerase chain reaction (RT-PCR). Phase separation One embodiment of the invention provides methods and reagents to enhance the purity of isolated RNA by performing phenol extraction of an RNA-containing sample at a pH below 4.0. In one embodiment, the pH ranges from about pH 3.9 to about pH 3.6. Phenol extraction at a pH below 4.0 more effectively separates RNA from DNA than phenol extraction at pH 4.0 or higher. The RNA isolating reagent used in the inventive phase separation method comprises an aqueous solution of phenol, and a buffer to maintain the pH within the range from about 3.6 to below pH 4.0. In one embodiment, the pH ranges between pH 3.7 to pH 3.9. The effective concentration of phenol in the RNA isolating reagent ranges from about 10%w/w to about 60%w/w. In one embodiment, the concentration of phenol ranges from about 25%w/w to about 45%w/w. The composition may also include other components, such as inhibitors of ribonuclease (RNase), salts, chelating agents, solubilizing agents, detergents, chaotropes, and phenol derivatives. In some embodiments, RNA in samples having low RNase activity, such as cultured cells, may be extracted with acidic phenol at a pH between about 3.6 to below pH 4.0, and this may sufficiently protect against RNA degradation. However, phenol may not adequately prevent degradation of RNA by cellular RNases derived from the sample or from contaminated labware. Thus, an effective amount of at least one RNase inhibitor may be included in the composition. The RNase inhibitor may be present during sample homogenization and/or during acid phenol extraction. RNase inhibitors include proteinase K, ribonuclease inhibitor from human placenta, vanadyl ribonucleoside complex, and chaotropic salts. Chaotropic salts include guanidine thiocyanate, guanidine hydrochloride, and mixtures of these. In one embodiment, an effective concentration of chaotropic salts ranges from about 0.5 M to about 6 M. In another embodiment, an effective concentration of chaotropic salts ranges from about 2 M to about 4 M. The buffer may be salts of at least one of acetate, citrate, phosphate, phthalate, tartrate, or lactate. The concentration of buffer should be sufficient to maintain the composition at a pH between about 3.6 to below 4.0. In one embodiment, the pH ranges from about 3.75 to about 3.85. The buffer may be added before or after sample homogenization, either separately or together with the phase separation reagent. Some samples with a high buffering capacity, such as blood and plant tissues, may require an additional amount of acid to adjust the pH within the desired range. The inventive composition may also contain organic and inorganic salts such as chloride, phosphate, acetate and thiocyanate salts of sodium, potassium, lithium and ammonium. The inventive composition may contain chelating agents such as citrates and ethylenediamine tetraacetate salts. The inventive composition may contain detergents such as polyoxyethylenesorbitan, sodium dodecylsulfate and sarcosine. The salts, chelating agents, and detergents promote tissue solubilization and precipitation of substantially pure RNA. To assist in solubilizing phenol, the aqueous composition may contain a solubilizer or mix of solubilizers. Solubilizers include polyalcohols such as glycerol at a concentration from about 1%w/w to about 10%w/w, the upper limit selected so as not to increase DNA contamination of the isolated RNA. Solubilizers also include guanidine salts. The inventive composition may contain within the about 60%w/w phenol, up to about 5%w/w of phenol derivatives that are less toxic than phenol itself. These derivatives include phenylethanol, propylene phenoxytol, thymol, or butylphenol. In one embodiment these derivatives are present in an amount ranging between about 1 %w/w to about 5%w/w. The composition may also contain insoluble or partially water-soluble organic compounds, such as cyclohexanol, cyclohexyl bromide, and dichlorobenzoic acid. These compounds increase the density of the composition and substitute for phenol, thereby minimizing the toxicity of the composition. In one embodiment of the phase separation method, a sample is prepared, typically by homogenization or lysis, in the inventive composition. The bulk of DNA and particulate matter may be removed by sedimentation or filtration from the homogenate or lysate. The homogenate or lysate is separated into aqueous and organic phases by mixing with a hydrophobic organic solvent or mix of solvents, such as chloroform, carbon tetrachloride, bromonaphtalene, bromoanisole or bromochloropropane. The mixture may be sedimented by centrifugation, for example, centhfugation at a temperature in the range between about 40C to about 10°C. The top aqueous phase contains RNA, and the interphase and organic phase contains DNA and proteins. RNA is precipitated from the aqueous phase with a water-soluble organic solvent, such as a lower alcohol. The precipitated RNA is washed by sedimentation or filtration and solubilized in water, formamide, or a buffer. The final RNA preparation is substantially pure, that is, it is undegraded and is essentially free of DNA contamination when tested by RT-PCR. Additionally, the inventive phase separation method is compatible with the method for the simultaneous isolation of RNA, DNA, and proteins. The DNA and proteins sequestered into the interphase and organic phase may be recovered, as described in Chomczynski, 1993; TRI Reagent brochure, 2003. Alternatively, DNA is precipitated from the organic phase and interphase by adding 0.3 volume of ethanol, followed by precipitation of proteins with a higher amount of ethanol. For example, DNA can be re-extracted from the interphase and organic phase with an aqueous solution at pH 7.0 or higher. Re-extracted DNA is precipitated from the aqueous solution with ethanol. As will be appreciated, the inventive composition and method may be used to isolate substantially pure RNA, substantially pure DNA (that is, DNA essentially free of RNA), and proteins from the same sample. Isolation of all three components allows for correlation of gene expression patterns with changes in the DNA sequence and protein content in biological samples, as well as having numerous other applications. In one embodiment, the composition used for homogenizing or lysing the sample may lack one or more components, which would be thereafter added to the homogenate or lysate, either alone or together with the phase separation solvent (for example chloroform). In another embodiment, sample homogenization or lysis may be performed above pH 4.0, in which case an acid or a buffer is then added to the homogenized or lysed sample in an amount sufficient to bring the pH of the homogenate or lysate within the range between about 3.6 to below pH 4.0. This amount of acid or buffer may be directly added to the homogenate or lysate, or it may be dissolved in the phase separation solvent. When added together with the phase separation solvent, the acid may be formic acid, acetic acid, trichloroacetic acid, aminocaproic acid, lactic acid, or chlorophenylacetic acid. To promote acid solubility, the phase separation solvent may contain solubilizers such as glycols. In one embodiment, sample homogenization or lysis is performed in a phenol-free solution containing an RNase inhibitor. After homogenization or lysis, phenol is added to achieve a final concentration ranging from about 10%w/w to about 60%w/w and extraction is performed at pH from about 3.6 to below 4.0. This pH range during extraction is maintained by a buffer that may be part of the aqueous solution, or added to phenol, or may be added separately. After phase separation by centrifugation, RNA is precipitated from the aqueous phase with alcohol. The precipitated RNA is washed and may be dissolved in a solvent such as water, buffer or formamide. Acidic phenol precipitation of DNA Leaving RNA in Supernatant One embodiment of the invention isolates substantially pure RNA using an acidic phenol solution without performing phase separation. Certain concentrations of acidic phenol selectively precipitate DNA (both single stranded DNA (ssDNA) and double stranded DNA (dsDNA), proteins, and other cellular components, while RNA remains in a soluble form. This unexpected phenomenon was utilized to elaborate reagents and methods for isolating RNA without separating aqueous and organic phases and the interphase. The acidic phenol precipitation method simplifies the process of RNA isolation. It also eliminates toxic organic solvents that may be used in the phase separation method. The composition propels DNA and proteins to form a firm pellet at the bottom of a tube, which alleviates the danger of accidental transfer of DNA and protein molecules to the supernatant fraction containing RNA. The supernatant can be securely collected by pipetting, siphoning, decanting, or filtering, each of which may be automated for use in an automated procedure for RNA isolation. Additionally, the entire acidic phenol precipitation method may occur at room temperature, which eliminates the need for a refrigerated centrifuge that may be used in the phase separation method. The composition used for acidic phenol precipitation comprises an aqueous solution of phenol at a concentration ranging from about 3%w/w to less than 30%w/w. In one embodiment, the phenol concentration ranges from about 3%w/w to about 25%w/w. In another embodiment, the phenol concentration is in the range between about 8%w/w to about 20%w/w. The phenol concentrations in the acid precipitation embodiment are lower than the 30%w/w phenol to 60%w/w phenol concentrations described in the '155 and '994 patents. The inventive composition is acidified with a buffer or an acid in an amount sufficient to maintain the pH within a range from about 3.6 to about 5.5. In one embodiment, the pH ranges from about 3.9 to about 4.5. The buffer can be selected from organic or inorganic buffers including, but not limited to, acetate, citrate, phosphate, phthalate, tartrate, and/or lactate. To enhance the efficiency of RNA isolation, acidic phenol may be supplemented with RNase inhibitors, salts, chelating agents, phenol solubilizing agents and/or detergents. RNase inhibitors include vanadyl ribonucleoside complex and protinase K or combinations of these inhibitors. RNase inhibitors also include chaotropic agents or chaotropes such as guanidine salts at concentrations ranging from about 0.5 M to about 6 M. In one embodiment, the concentration of the chaotropes is from about 1.5 M to about 2.5 M. Chaotropic salts may serve as phenol solubilizers by maintaining phenol in aqueous solution. The acidic phenol composition may further contain organic and/or inorganic salts such as chloride, phosphate, acetate, citrate and thiocyanate salts of sodium, potassium, lithium, and ammonium. The composition may also contain chelating agents such as citrates and ethylenediamine tetraacetate salts. The composition may also contain detergents including polyoxyethylenesorbitan, sodium dodecylsulfate, and sarcosine. The composition may also contain up to 5%w/w of solvents and reagents that are less toxic than phenol, such as thymol, phenylethanol, cyclohexanol, cyclohexyl bromide and dichlorobenzoic acid. These additional components promote tissue solubilization and precipitation of pure RNA. The acidic phenol precipitation reagent may contain an additional solubilizer or mix of solubilizers to help maintain phenol in aqueous solution. Phenol solubilizing agents include glycols, polyalcohols, and lower alcohols. These can be added to the acidic phenol precipitation reagent in amounts from about 1%w/w to about 10%w/w, the upper limit selected so as not to increase DNA contamination of the isolated RNA. In one embodiment of the acidic phenol precipitation method, a biological sample is homogenized or lysed in the precipitation reagent. The resulted homogenate or lysate is centrifuged or filtered to remove precipitated DNA, proteins, and other cellular components. RNA remains in a soluble form and is subsequently precipitated from the supernatant with a water- soluble organic solvent, such as lower alcohols including methanol, ethanol, propanol, isopropanol, and butanol. The RNA pellet is then washed and may be dissolved in water, buffer, or formamide. In the another embodiment of the acidic phenol precipitation method, a biological sample is homogenized in about 3 times (3X) to about 1.5 times (1.5X) concentrated acidic phenol precipitation reagent. The use of a concentrated reagent allows processing of solid tissues as well as high volume liquid samples using one reagent. A high volume of a liquid sample can be compensated by adding to the concentrated reagent a smaller amount of water. The concentrated reagent dissolves most of the components in a biological sample and effectively releases RNA from cellular structures. For example, a sample may be homogenized in two times (2X) concentrated reagent. Following homogenization, an equal volume of water is mixed with the homogenate. The addition of water brings phenol, guanidine, and other ingredients within the desired concentration. This creates conditions for effectively precipitating and removing DNA and proteins from an RNA containing sample. After centrifugation or filtration of the homogenate or lysate, RNA remains in the supernatant or filtrate, while DNA, protein, and other cellular components form a firm pellet at the bottom of a tube. RNA is precipitated from the supernatant or filtrate with a water-soluble organic solvent as previously described. The RNA precipitate is washed and may be dissolved in water, buffer, or formamide. In one embodiment of the invention, a single reagent may be used in either the phase separation method or the acidic phenol precipitation method. This dual use reagent comprises components of the reagent used in the phase separation method and a 2X concentration of reagent used in the acidic phenol precipitation method. As previously described, the pH for the phase separation method may be between about pH 3.6 to below pH 4.0, and the pH for the acidic phenol precipitation method may be between about pH 3.6 to about pH 5.5. In the dual use embodiment, a pH adjustment of the reagent is therefore necessary before switching from one method to the other method. For example, the 2X reagent for the acidic phenol precipitatioh method at pH 4.2 must be further acidified to a pH below 4.0 before use in the phase separation method. The inventive phase separation method may be used for specimens containing high amounts of fats, such as fat tissue and certain tumor or neoplastic tissues. Samples with a high level of contaminants, such as plants and fat- containing tissues, may be processed by the dual use procedure, which combines the acidic phenol precipitation method and the phase separation method. In one embodiment, a biological sample is homogenized in 2X acidic phenol precipitation reagent. The homogenate is then diluted with water to approach the concentration range of the acidic phenol precipitation reagent (that is, about 3%w/w phenol to less than 30%w/w phenol). After dilution, precipitated DNA, proteins and other cellular components are removed by sedimentation or filtration. The resulting supernatant or filtrate is collected and mixed with a phase separation solvent. In one embodiment, 0.05 volume to 0.01 volume of a phase separation solvent is added per one volume of the supernatant. The phase separation solvent or mix of solvents is at least one hydrophobic solvent including, but not limited to, caprolactone, ethylene glycol diacetate, polyethylene glycol dibenzoate, as well as solvents used for the phase separation method. The mixture is centrifuged to obtain a top aqueous phase, an interphase, and an organic phase. The aqueous phase containing RNA is collected and mixed with one volume of a lower alcohol to precipitate RNA. The precipitated RNA is washed and dissolved in water, buffer, or formamide. The methods providing purified RNA based on the inventive acidic phenol solutions are useful for gene expression profiling with RT-PCR based microarrays used in biotechnology, molecular biology and clinical applications. This can be exemplified by the detection of specific gene expression patterns in cancer cells and other type of pathological specimens. Selective RNA precipitation using low volume organic solvent As previously described, RNA may be precipitated from the aqueous phase in the inventive phase separation method, and from the inventive acidic phenol composition. In each case, RNA is precipitated by adding about one volume of an organic solvent to approach a final organic solvent concentration of about 50%w/w. However, in some sample preparations, one volume of organic solvent co-precipitates polysaccharides and proteins, such as proteoglycans, together with RNA. Previously, to avoid co-precipitation of contaminants, one method modified the single-step RNA purification method by employing 25%w/w alcohol in the presence of 0.9 M sodium ions to precipitate RNA (Chomczynski, 1995). Another method treated a phenol-free chaotrope solution with 13%w/w to 23%w/w of an organic solvent, with the pH of the solution remaining within the range of pH 6 to pH 7.5 to precipitate RNA. In the present inventive method, substantially pure RNA is precipitated from phenol-chaotrope solutions by adding an organic solvent or mix of solvents to achieve a final concentration of about 10%w/w to about 40%w/w organic solvent. The organic solvent(s) may be acetone, tetramethylene sulfone, lower alcohols, glycols, polyalcohols, acetone, ethyleneglycol diacetate, and/or methyl sulfoxide. This method provides substantially pure RNA when precipitating RNA from either the aqueous phase in the phase separation method, or in the acidic phenol precipitation method from the DNA- and protein-free supernatant. The method does not require adding salts to a phenol-chaotrope solution. It was unexpected that substantially pure RNA precipitated from a phenol- chaotrope solution at about 10%w/w to about 40%w/w concentration of an organic solvent without supplementing the solution with salt, as was earlier suggested (Chomczynski 1995). In fact, adding salt along with alcohol decreased the purity of the isolated RNA. The finding that 10%w/w to 40%w/w alcohol alone precipitated RNA from the phenol-chaotrope solution was also contrary to a report where DNA precipitated and RNA remained in a soluble form by adding 0.3 volume of alcohol (final concentration 24%w/w) to the phenol-chaotrope solution (Siebert, 1993). In one embodiment, the final concentration of organic solvent(s) in the composition is from about 20%w/w to about 25%w/w. In another embodiment, the final concentration of organic solvent(s) in the composition is from about 10% to about 40%. The pH of the phenol-chaotrope solution may range from about pH 2.0 to about pH 9.0. In one embodiment, the pH of the phenol-chaotrope solution ranges from about pH 3.5 to about pH 5.0. Organic solvents at concentrations from about 10%w/w to about 40%w/w precipitate RNA molecules greater than about 200 bases, considered as higher molecular weight RNA. RNA fragments less than about 200 bases, along with polysaccharides and proteoglycans, remain in solution. Following precipitation of higher molecular weight RNA, the smaller molecular weight RNA can be recovered from the solution by precipitating with an additional amount of organic solvent to approach a final concentration of about 50%w/w or higher, for example, to about 90%w/w. The inventive method, whereby RNA is precipitated using about 10%w/w to about 40%w/w of an organic solvent, can also be used to decrease the amount of contaminating DNA in RNA preparations. This selective precipitation of RNA is effective only when a small amount of contaminating DNA is present in the solutions, for example, less than 10 ng DNA per 1 μg RNA. Precipitating RNA using about 10%w/w to about 40%w/w of an organic solvent also improves the quality of RNA isolated, such that a high yield of substantially pure and undegraded RNA is obtained, in accord with the method described in my previous '155 and '994 patents. Acidic RNA Precipitation from Salt-Containing Solutions Substantially pure RNA, along with DNA, may be obtained from aqueous solutions containing salts by pH-dependent precipitation of RNA at a pH below about 3.3. Precipitating RNA from salt solutions at an acidic pH is contrary to that reported in U.S. Patent No. 5,973,137. The '137 patent discloses that, in solutions at pH below 6, non-chaotropic salts precipitate DNA, while RNA stays in a soluble form. In the inventive method, a buffer acid is added to the RNA solution in an amount sufficient to result in a pH of 3.3 or lower. In one embodiment, the resulting pH is in the range from about pH 3.0 to about pH 2.7. The acid may be an organic acid or an inorganic acid. The acid may be hydrochloric acid, phosphoric acid, acetic acid, and/or lactic acid. In one embodiment, salts in the composition may be guanidine salts. This embodiment may be incorporated into either the inventive phase separation method and/or the inventive acidic phenol precipitation method. For use in the acidic phenol precipitation method, acids or buffers can be dissolved either in water or in organic solvents. The acid selectively precipitates RNA, leaving polysaccharides and protein in a soluble form. The volume of acid used to precipitate RNA is small, permitting a low sample volume during RNA isolation. In one embodiment, the volume of acid ranges from about 0.1%w/w to about 25%w/w of the volume of RNA solution. The precipitation of RNA with a low amount of an organic solvent and with acidic pH, described in the present invention, can also be used to improve the quality of RNA using methods disclosed in my previous '155 and '994 patents. Treating an RNA-containing sample obtained by RNA precipitation with a low volume of an organic solvent or acidic precipitation of RNA from salt-containing solutions precipitates higher molecular weight RNA. Higher molecular weight RNA includes ribosomal RNA (rRNA, for example 18S and 28S RNA) and messenger RNA (mRNA). The remaining lower molecular weight RNA is less than about 200 nucleotides, and includes transfer RNA (tRNA), 5S RNA, and small interfering RNA (siRNA) that regulates gene expression. Lower molecular weight RNA is recovered from the above-described solution by treatment with an additional volume of an organic solvent. In one embodiment, the sample is treated with one volume of a lower alcohol, for example, methanol, ethanol, propanol, etc. to precipitate low molecular weight RNA. Exemplary solutions and methods of the present invention are described in the following working Examples. Example 1 Phase separation isolation of RNA from rat liver In one embodiment, the following composition was used for phase separation of RNA: 4 M guanidine thiocyanate, 0.2 M ammonium thiocyanate, 5%w/w glycerol, 40%w/w phenol, 0.1%w/w sarcosine, 10 mM sodium citrate, and 0.1 M sodium acetate buffer, pH 3.8. Rat liver (38 mg) was homogenized in 1.5 ml of the above composition. Thereafter, and sedimented for fifteen minutes at 4°C at 12,000 x g. Following sedimentation, an aqueous phase, an interphase, and a lower organic phase formed. RNA sequestered into the aqueous phase, while DNA and proteins sequestered into the interphase and organic phase. RNA was precipitated from the aqueous phase by adding 0.75 ml of isopropanol. The RNA precipitate was centrifuged for five minutes at 10,000 x g. The resulting pellet was washed with 0.75 ml of 75%w/w ethanol and centrifuged for five minutes at 10,000 x g. The final RNA pellet was dissolved in water and the RNA concentration was determined spectrophotometrically at A26o/2βo by methods known to one skilled in the art. The yield of RNA was 0.22 mg. The A26o/2βo ratio was 1.76, which indicated the lack of protein contamination. The isolated RNA was successfully utilized for RT-PCR using primers for glyceraldehydes 3-phosphate dehydrogenase (GAPDH),. actin, and c-fos genes. Reverse transcription was performed using Superscript transcriptase from Invitrogen (Carlsbad CA) and PCR was performed using Taq DNA polymerase from Sigma (St. Louis MO). RT-PCR products were analyzed on a 1% agarose-ethidium bromide gel. No DNA was detected in the isolated RNA in the absence of reverse transcription. Northern blot analysis of the isolated RNA was performed using 1%-formaldehyde-agarose gels and transferred to a nylon membrane. Ethidium bromide and methylene blue staining showed undegraded ribosomal bands. Hybridization with biotin-labeled probes showed undegraded bands of mRNA for GAPDH, actin, and c-fos. Example 2 Phase separation isolation of RNA from human blood Human blood (0.5 ml) was mixed with 75 μl of glacial acetic acid and 5 ml of the composition described in Example 1. Thereafter, 0.5 ml of bromochloropropane was added to the mixture. The mixture was shaken and sedimented for fifteen minutes at 4°C at 12,000 x g. Following sedimentation, the mixture formed an aqueous phase, an interphase, and a lower organic phase. RNA sequestered into the aqueous phase, while DNA and proteins sequestered into the interphase and organic phase. RNA was precipitated from the aqueous phase by adding 1.25 ml of isopropanol. The RNA precipitate was centrifuged for five minutes at 12,000 x g. The resulting pellet was

-15- washed with five ml of 75% ethanol and ceπtrifuged for five minutes at 10,000 x g. The final RNA pellet was dissolved in water and the RNA concentration was determined spectrophotometrically at A26o/28o by methods known to one skilled in the art. The yield of RNA was 18.9 μg. The A26o/28o ratio was 1.70, which indicated the lack of protein contamination. The isolated RNA was successfully utilized for RT-PCR using GAPDH primers. No DNA contamination was detected by PCR of the isolated RNA without reverse transcription. Northern blot analysis of the isolated RNA showed undegraded bands of ribosomal RNA and an undegraded band of GAPDH mRNA. Example 3 Isolation of RNA by phase separation and acidified bromochloropropane Rat spleen (21 mg) was homogenized in 1 ml of an aqueous solution containing 3.5 M guanidine thiocyanate, 50 mM potassium acetate, 43%w/w phenol, 0.1 % Triton X-100, pH 4.1. The homogenate was centrifuged at 12,000 x g for ten minutes to remove the bulk of DNA and particulates. The clear homogenate was mixed with 0.1 ml of bromochloropropane containing 14%w/w acetic acid. The resulting pH of the mixture was pH 3.7. The mixture was shaken and sedimented for ten minutes at 4°C at 12,000 x g. Following sedimentation, the mixture formed an aqueous phase, an interphase, and a lower organic phase. RNA sequestered into the aqueous phase, while DNA and proteins sequestered into the interphase and organic phase. RNA was selectively precipitated from the aqueous phase by adding 0.5 ml ethanol. The precipitated RNA was sedimented for five minutes at 10,000 x g, then washed with 75%w/w ethanol and sedimented for five minutes at 10,000 x g. The final RNA pellet was dissolved in water and the RNA concentration was determined spectrophotometrically at A26o/28o by methods known to one skilled in the art. The yield of RNA was 77 μl. The A26o/28o ratio was 1.74, which indicated the lack of protein contamination. The isolated RNA was successfully utilized for RT-PCR using GAPDH primers and no DNA contamination was detected. Northern blot analysis of the isolated RNA showed undegraded bands of ribosomal RNA and an undegraded band of GAPDH mRNA. Example 4 Isolation of RNA by phase separation and homogenization in a phenol-free chaotrope solution Rat skeletal muscle (29 mg) was homogenized in 0.5 ml of an aqueous solution of 3 M guanidine thiocyanate and 5 mM sodium acetate. The homogenate was mixed with 0.5 ml of phenol and 0.1 M sodium acetate buffer, pH 3.7. The resulting mixture was shaken with 0.1 ml of bromochloropropane and sedimented for fifteen minutes at 40C at 12,000 x g. Following sedimentation, the mixture formed an aqueous phase, an interphase, and a lower organic phase. RNA sequestered into the aqueous phase, while DNA and proteins sequestered into the interphase and organic phase. RNA was selectively precipitated from the aqueous phase by adding 0.5 ml of an aqueous solution containing 50%w/w ethanol. The RNA precipitate was washed, treated, and assayed as described in Example 1. The yield of RNA was 16 μg. The A26o/28o ratio was 1.70, which indicated the lack of protein contamination. The isolated RNA was successfully utilized for RT-PCR using GAPDH primers and no DNA contamination was detected. Northern blot analysis of the isolated RNA showed undegraded bands of ribosomal RNA and an undegraded band of GAPDH mRNA. Example 5 Isolation of RNA by phase separation with homogenization in 1%w/w sodium dodecyl sulfate A primary culture of human fibroblast cells (Clonetics, San Diego CA) grown in a 25 cm2 plastic bottle was overlaid with 1.5 ml of a solution containing 1%w/w sodium dodecyl sulfate and 10 mM sodium citrate, pH 7.0, supplemented with 50 μg/ml proteinase K. The resulting cell solution was incubated for one hour at room temperature (about 200C), transferred to a centrifuge tube, and mixed with 1.5 ml of acidic phenol containing 12%w/w water and 100 mM sodium acetate, pH 3.7. After centrifugation for fifteen minutes at 4°C, the mixture formed an aqueous phase, an interphase, and an organic phase. Following phase separation, RNA sequestered into the aqueous phase, while DNA and proteins sequestered into the interphase and organic phase, respectively. RNA was precipitated from the aqueous phase by adding 0.75 ml of ethanol and sedimenting for five minutes at 10,000 x g. The RNA pellet was washed with 75% ethanol, centrifuged for five minutes at 10,000 x g, and dissolved in water. The yield of RNA was 18 μg. The A26o/2βo ratio was 1.71 , which indicated the lack of protein contamination. The isolated RNA was successfully utilized for RT-PCR using GAPDH primers and no DNA contamination was detected. Northern blot analysis of the isolated RNA showed undegraded bands of ribosomal RNA and an undegraded band of GAPDH mRNA. Example 6 Isolation of RNA by acidic phenol precipitation In one embodiment, the following composition was used for acidic phenol precipitation of RNA: 20%w/w phenol, 2 M guanidine thiocyanate, 15 mM sodium citrate, 0.1 M lithium chloride, 0.05%w/w sarcosine, 1.5%w/w glycerol, and sodium acetate buffer, pH 4.2. Rat liver (52 mg) was homogenized in 1 ml of this composition. The homogenate was centrifuged at 10,000 x g for five minutes at room temperature (about 200C) to remove precipitated DNA, protein, and cellular components. The resulting supernatant was transferred to a clean tube and was mixed with 1 ml of ethanol to precipitate RNA. Precipitated RNA was sedimented at 10,000 x g for five minutes, washed with 75%w/w ethanol, and dissolved in water. The yield of RNA was 187 μg. The A26o/2βo ratio was 1.74, which indicated the lack of protein contamination. The isolated RNA was successfully utilized for RT-PCR using GAPDH primers and no DNA contamination was detected. Northern blot analysis of the isolated RNA showed undegraded bands of ribosomal RNA and an undegraded band of GAPDH mRNA. Example 7 Isolation of RNA by acidic phenol precipitation using two times concentrated reagent Rat liver (47 mg) was homogenized in 1 ml of the reagent described in Example 6 at two times the concentration indicated in Example 6. The concentrated reagent contained additionally 1 %w/w phenylethanol. The homogenate was mixed with 1 ml water to form the precipitating reagent. The precipitated DNA, proteins and other cellular components were sedimented by centrifugation at 10,000 x g for five minutes at room temperature (about 2O0C). The resulting supernatant was transferred to a clean tube and mixed with 1 ml ethanol to precipitate RNA. Precipitated RNA was sedimented at 10,000 x g for 5 minutes, washed with 75%w/w ethanol, and dissolved in water. The yield of RNA was 178 μg. The A26o/2so ratio was 1.77, which indicated the lack of protein contamination. The isolated RNA was successfully utilized for RT-PCR using GAPDH primers and no DNA contamination was detected. Northern blot analysis of the isolated RNA showed undegraded bands of ribosomal RNA and an undegraded band of GAPDH mRNA. Example 8 Isolation of RNA by the inventive two-step procedure Rat brain (61 mg) was homogenized in 1 ml of the two times concentrated reagent described in Example 7. The homogenate was mixed with 1 mi water and the resulting mixture was centrifuged at 10,000 x g for five minutes at room temperature (about 200C) to remove precipitated DNA, protein, and cellular components. The supernatant was transferred to a clean tube and mixed with 0.05 ml of bromochloropropane. The mixture was centrifuged and separated into a top aqueous phase, an interphase, and an organic phase. The aqueous phase containing RNA was transferred to a fresh tube and was acidified to pH 2.9 with 5 M lactic acid in isopropanol. The RNA precipitate was sedimented at 10,000 x g for five minutes, washed with 75%w/w ethanol, and dissolved in water. The yield of RNA was 33 μg. The A2so/2βo ratio was 1.77, which indicated the lack of protein contamination. The isolated RNA was successfully utilized for RT-PCR using GAPDH primers and no DNA contamination was detected. Northern blot analysis of the isolated RNA showed undegraded bands of ribosomal RNA and an undegraded band of GAPDH mRNA. Other variations or embodiments of the invention will also be apparent to one of ordinary skill in the art from the above description and examples. Thus, the forgoing embodiments are not to be construed as limiting the scope of this invention.

1. A phenol-containing composition for isolating purified RNA comprising phenol at a final concentration ranging from about 3%w/w to about 98%w/w and a buffer sufficient to maintain a pH of the final composition in the range from pH 3.6 to below pH 4.0.

2. A phenol-containing composition for isolating purified RNA comprising phenol at a final concentration ranging from 3%w/w to less than 30%w/w, and a buffer sufficient to maintain a pH of the final composition in the range from pH 3.9 to pH 5.5.

3. The composition according to claims 1 or 2 where the buffer is selected from at least one of acetate, citrate, phosphate, phthalate, tartrate, lactate, or mixtures thereof.

4. The composition according to claims 1 or 2 further comprising at least one ribonuclease inhibitor.

5. The composition according to claims 1 or 2 further comprising phenol derivatives selected from at least one of phenylethanol, propylene phenoxytol, thymol, butylphenol, or mixtures thereof at a final concentration up to 5%w/w.

6. The composition according to claims 1 or 2 further comprising phenol solubilizers selected from at least one of polyalcohols, monoalcohols, and guanidine salts.

7. The composition of claim 1 further comprising at least one organic compound in a concentration ranging from 1%w/w to 5%w/w sufficient to increase the density of the composition.

8. A phenol-free aqueous composition for isolating purified RNA, the phenol-free composition comprising at least one ribonuclease inhibitor and a buffer selected from at least one of acetate, citrate, phosphate, phthalate, tartrate, lactate, or mixtures thereof, sufficient to maintain a pH of the composition in the range from pH 3.6 to below pH 4.0.

9. The composition of any one of claims 1, 2, or 8 further comprising a detergent. 10. The composition of any one of claims 1, 2, or 8 further comprising an inorganic or organic salt and a chelating agent.

11. A phenol-free phase separation composition for use in isolating purified RNA by phase separation comprising at least one hydrophobic organic solvent at a final concentration in the range from 10%w/w to 40%w/w, and at least one acid sufficient to maintain a pH in the range of pH 3.6 to below pH 4.0 during phase separation, and an optional acid solubilizer.

12. The composition of claim 11 wherein the organic solvent is at least one of chloroform, carbon tetrachloride, bromochloropropane, bromonaphtalene, or bromoanisole.

13. The composition of claim 11 wherein the acid is at least one of formic acid, acetic acid, trichloroacetic acid, aminocaproic acid, lactic acid, or chlorophenylacetic acid.

14. A method for isolating purified RNA from a biological sample comprising a) treating the sample with a reagent comprising phenol at a final concentration ranging from 10%w/w to 60%w/w and at least one ribonuclease inhibitor, b) mixing the sample with at least one hydrophobic solvent while maintaining a pH in the range from pH 3.6 to below pH 4.0, c) recovering purified RNA from an aqueous phase to which about an equal volume of a water-soluble organic solvent is added to precipitate the purified RNA, and d) washing and solubilizing the precipitated RNA.

15. The method of claim 14 wherein the reagent in (a) further comprises a buffer selected from at least one of acetate, citrate, phosphate, phthalate, tartrate, lactate, or mixtures thereof.

16. The method of claim 14 wherein the reagent in (a) further comprises at least one ribonuclease inhibitor. 17. The method of claim 14 wherein the reagent in (a) further comprises a detergent.

18. The method of claim 14 wherein the reagent in (a) further comprises an inorganic or organic salt and a chelating agent.

19. The method of claim 14 wherein the reagent in (a) further comprises phenol derivatives selected from at least one of phenylethanol, propylene phenoxytol, thymol, butylphenol, or mixtures thereof at a final concentration up to 5%w/w.

20. The method of claim 14 wherein the reagent in (a) further comprises phenol solubilizers selected from at least one of polyalcohols, monoalcohols, and guanidine salts.

21. The method of claim 14 wherein the reagent in (a) further comprises at least one organic compound in a concentration ranging from 1 %w/w to 5%w/w sufficient to increase the density of the composition.

22. The method of claim 14 wherein the sample is first treated with the phenol-free composition of either of claims 8 or 11 before step (a).

23. The method according to claims 14 or 20 wherein the reagent in (a) is buffered to maintain a pH in the range from pH 3.6 to below pH 4.0.

24. The method according to claims 14 or 22 wherein step (a) is performed at a pH ranging from pH 3.9 to pH 9.0, and the sample is then adjusted to a pH ranging from pH 3.6 to below pH 4.0. 25. A method for isolating purified RNA from a biological sample comprising the steps of a) treating the sample with a reagent comprising phenol at a final concentration ranging from 3%w/w to less than 30%w/w and a buffer sufficient to maintain a pH of the composition in the range from pH 3.6 to pH 5.5, b) sedimenting or filtering the sample to obtain a purified sample substantially free of DNA, proteins, and cellular components, adding to the purified sample an equal volume of a water-soluble organic solvent to precipitate purified RNA, d) sedimenting or filtering the precipitated RNA, and e) washing and solubilizing the precipitated RNA.

26. A two-step method for isolating purified RNA from a biological sample comprising a) treating the sample with a reagent comprising phenol at a final concentration ranging from 3%w/w to less than 30%w/w, at least one chaotrope, and a buffer sufficient to maintain a pH of the composition in the range from pH 3.6 to pH 5.5, b) sedimenting or filtering the sample to obtain a purified sample substantially free of DNA, proteins, and cellular components, c) adding to the purified sample at least one hydrophobic organic solvent and a buffer in a concentration sufficient to maintain a pH of the purified sample in the range from pH 3.6 to below pH 4.0, d) recovering purified RNA from an aqueous phase to which an equal volume of a water soluble organic solvent is added to precipitate purified RNA, e) sedimenting or filtrating the precipitated RNA, and f) washing and solubilizing the precipitated RNA. 27. The method of claim 26 where the hydrophobic organic solvent is sufficiently dense to separate the organic phase during phase separation.

28. The method according to any one of claims 25 or 26 wherein the hydrophobic organic solvent is selected from at least one of caprolactone, ethylene glycol diacetate, polyethylene glycol dibenzoate, chloroform, carbon tetrachloride, bromochloropropane, bromonaphtalene, bromoanisole, or mixtures thereof.

29. The method according to any one of claims 25 or 26 wherein the sample is treated with the composition of (a) at 1.5X to 2.5X concentration, and the resulting sample is diluted to approach the non-concentrated solution.

30. The method according to any one of claims 14, 22, 25, or 26 wherein the solvent added to precipitate RNA is at least one of lower alcohols, polyalcohols, acetone, ethyleneglycol diacetate, methyl sulfoxide, or mixtures thereof.

31. A method for isolating purified RNA from an RNA- and salt-containing solution comprising adjusting a pH of the solution with a buffer in an amount sufficient to result in a maximum pH of 3.3, thereafter precipitating the purified RNA.

32. The method of claim 31 wherein the pH ranges from pH 3.0 to pH 2.7.

33. The method of claim 31 wherein the buffer is at least one of an organic acid or an inorganic acid.

34. The method of claim 31 wherein the salt is selected from the group consisting of sodium, potassium, lithium and guanidine salts.

35. The method of claim 31 wherein the solution contains phenol at a concentration from 1 %w/w to 60%w/w. 36. A method for selectively precipitating higher molecular weight RNA from a biological sample comprising treating the sample with an aqueous composition comprising phenol at a final concentration ranging from 1%w/w to 60%w/w, at least one chaotrope, a buffer in a concentration sufficient to maintain a pH of the composition in the range from pH 2.0 to pH 9.0, at least one water- soluble organic solvent at a concentration from 10%w/w to 40%w/w to selectively precipitate higher molecular weight RNA from the sample, and precipitating purified higher molecular weight RNA from the sample.

37. The method of claim 36 further comprising the step of thereafter adding additional organic solvent sufficient to increase the concentration of organic solvent to at least 50%w/w to precipitate lower molecular weight RNA, and precipitating purified lower molecular weight RNA from the sample.

38. The method of claim 36 comprising preparing the biological sample according to any one of claims 14, 22, 25, or 26 to obtain an aqueous solution of RNA, and precipitating RNA from the aqueous solution. AMENDED CLAIMS received by the International Bureau on 29 September 2005 (29.09.2005): original claims 1, 2, 11-14, 25 and 26 have been amended. Original claims 3-10, 15-24, and 27-38 remain unchanged. 1. A pheπol-contalnlng mono-phase composition for isolating purified RNA comprising phenol at a final concentration ranging from 3%w/w to 99%w/w and a buffer sufficient to maintain a pH of the final composition in the range from pH 3,6 to below pH 4.0.

2. A phenot-coπtaiπing mono-phase composition for isolating purified RNA comprising phenol at a final concentration ranging from 3%w/w to less than 30%w/w, and a buffer sufficient to maintain a pH of the final composition in the range from pH 3.9 to pH 5.5,

3. The composition according to claims 1 or 2 where the buffer is selected from at least one of acetate, citrate, phosphate, phthalate, tartrate, lactate, or mixtures thereof.

4. The composition according to claims 1 or 2 further comprising at least one riboπuclease inhibitor.

5. The composition according to claims 1 or 2 further comprising phenol derivatives selected from at least one of phenylethanol, propylene phenoxytol, thymol, butylphenol, or mixtures thereof at a final concentration up to 5%w/w.

6. The composition according to claims 1 or 2 further comprising phenol solubllizers selected from at least one of polyalcohols, monoalcohols, and guaπidine salts.

7. The composition of claim 1 further comprising at least one organic compound in a concentration ranging from 1%w/w to 5%w/w sufficient to increase the density of the composition.

6. A phenol-free aqueous composition for isolating purified RNA, the phenol-free composition comprising at least one riboπuclease inhibitor and a buffer selected from at least one of acetate, citrate, phosphate, phthalate, tartrate, lactate, or mixtures thereof, sufficient to maintain a pH of the composition in the range from pH 3.6 to below pH 4.0.

9. The composition of any one of claims 1 , 2, or 8 further comprising a detergent.

26 10. The composition of any one of claims 1 , 2, or fi further comprising an inorganic or organic salt and a chelating agent,

11. CANCEL

12. CANCEL

13. CANCEL.

14. A method for isolating purified RNA from a biological sample comprising a) treating the sample with a reagent comprising phenol at a final concentration ranging from 10%w/w to 60%w/w and at least one rlbonuclease inhibitor, b) mixing the sample with at least one hydrophobic solvent while maintaining a pH in the range from pH 3.6 to below pH 4.0, c) recovering purified RNA from an aqueous phase to which an equal volume of a water- soluble organic solvent is added to precipitate the purified RNA, and d) washing and solubiliziπg the precipitated RNA.

15. The method of claim 14 wherein the reagent in (a) further comprises a buffer selected from at least one of acetate, citrate, phosphate, phthalate, tartrate, lactate, or mixtures thereof.

16. The method of claim 14 wherein the reagent in (a) further comprises at least one riboπudβasβ inhibitor. 25. An acidic phenol precipitation method for isolating purified RNA from a biological sample comprising the steps of a) treating the sample with a mono-phase reagent comprising phenol at a final concentration ranging from 3%w/w to less than 30%w/w and a buffer sufficient to maintain a pH of the composition in the range from pH 3.6 to pH 5.5, b) sedimenting or filtering the sample to obtain a purified sample substantially free of DNA1 proteins, and cellular components, c) adding to the purified sample an equal volume of a water-soluble organic solvent to precipitate purified RNA1 d) sedimenting or filtering the precipitated RNA, and e) washing and solubilizing the precipitated RNA.

26. A two-step method for isolating purified RNA from a biological sample comprising a) treating the sample with a mono-phase reagent comprising phenol at a final concentration ranging from 3%w/w to less than 30%w/w, at least one chaotrope, and a buffer sufficient to maintain a pH of the composition in the range from pH 3.6 to pH 5.5, b) sedimenting or filtering the sample to obtain a purified sample substantially free of DNA, proteins, and cellular components, c) adding to the purified sample at least one hydrophobic organic solvent and a buffer in a concentration sufficient to maintain a pH of the purified sample In the range from pH 3.6 to below pH 4.0, d) recovering purified RNA from an aqueous phase to which an equal volume of a water soluble organic solvent is added to precipitate purified RNA, e) sedimenting or filtrating the precipitated RNA, and f) washing and solubilizing the precipitated RNA.

RT-PCR: The Basics

2007-12-18T14:20:00.000-05:00

RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive technique for mRNA detection and quantitation currently available. Compared to the two other commonly used techniques for quantifying mRNA levels, Northern blot analysis and RNase protection assay, RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell.

This article first discusses the advantages of real-time RT-PCR compared to end-point methods. This discussion is followed by a description of the different methods for quantitating gene expression by real-time RT-PCR with respect to the different chemistries available, the quantitation methods used and the instrumentation options available. Subsequently, the “traditional” methods of quantitating gene expression by RT-PCR, i.e. end-point techniques, are presented.

Why Real-Time RT-PCR?

Over the last several years, the development of novel chemistries and instrumentation platforms enabling detection of PCR products on a real-time basis has led to widespread adoption of real-time RT-PCR as the method of choice for quantitating changes in gene expression. Furthermore, real-time RT-PCR has become the preferred method for validating results obtained from array analyses and other techniques that evaluate gene expression changes on a global scale.

To truly appreciate the benefits of real-time PCR, a review of PCR fundamentals is necessary. At the start of a PCR reaction, reagents are in excess, template and product are at low enough concentrations that product renaturation does not compete with primer binding, and amplification proceeds at a constant, exponential rate. The point at which the reaction rate ceases to be exponential and enters a linear phase of amplification is extremely variable, even among replicate samples, but it appears to be primarily due to product renaturation competing with primer binding (since adding more reagents or enzyme has little effect). At some later cycle the amplification rate drops to near zero (plateaus), and little more product is made.

For the sake of accuracy and precision, it is necessary to collect quantitative data at a point in which every sample is in the exponential phase of amplification (since it is only in this phase that amplification is extremely reproducible). Analysis of reactions during exponential phase at a given cycle number should theoretically provide several orders of magnitude of dynamic range. Rare targets will probably be below the limit of detection, while abundant targets will be past the exponential phase. In practice, a dynamic range of 2-3 logs can be quantitated during end-point relative RT-PCR. In order to extend this range, replicate reactions may be performed for a greater or lesser number of cycles, so that all of the samples can be analyzed in the exponential phase.

Real-time PCR automates this otherwise laborious process by quantitating reaction products for each sample in every cycle. The result is an amazingly broad 107-fold dynamic range, with no user intervention or replicates required. Data analysis, including standard curve generation and copy number calculation, is performed automatically. With increasing numbers of labs and core facilities acquiring the instrumentation required for real-time analysis, this technique is becoming the dominant RT-PCR-based quantitation technique.

Real-Time PCR Chemistries

Currently four different chemistries, TaqMan® (Applied Biosystems, Foster City, CA, USA), Molecular Beacons, Scorpions® and SYBR® Green (Molecular Probes), are available for real-time PCR. All of these chemistries allow detection of PCR products via the generation of a fluorescent signal. TaqMan probes, Molecular Beacons and Scorpions depend on Förster Resonance Energy Transfer (FRET) to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moeity to the same or different oligonucleotide substrates. SYBR Green is a fluorogenic dye that exhibits little fluorescence when in solution, but emits a strong fluorescent signal upon binding to double-stranded DNA.

TaqMan Probes

TaqMan probes depend on the 5'- nuclease activity of the DNA polymerase used for PCR to hydrolyze an oligonucleotide that is hybridized to the target amplicon. TaqMan probes are oligonucleotides that have a fluorescent reporter dye attached to the 5' end and a quencher moeity coupled to the 3' end. These probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5'- nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent and quenching dyes and FRET no longer occurs. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage

Well-designed TaqMan probes require very little optimization. In addition, they can be used for multiplex assays by designing each probe with a spectrally unique fluor/quench pair. However, TaqMan probes can be expensive to synthesize, with a separate probe needed for each mRNA target being analyzed.

Molecular Beacons

Like TaqMan probes, Molecular Beacons also use FRET to detect and quantitate the synthesized PCR product via a fluor coupled to the 5' end and a quench attached to the 3' end of an oligonucleotide substrate. Unlike TaqMan probes, Molecular Beacons are designed to remain intact during the amplification reaction, and must rebind to target in every cycle for signal measurement. Molecular Beacons form a stem-loop structure when free in solution. Thus, the close proximity of the fluor and quench molecules prevents the probe from fluorescing. When a Molecular Beacon hybridizes to a target, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation.

Molecular Beacons, like TaqMan probes, can be used for multiplex assays by using spectrally separated fluor/quench moieties on each probe. As with TaqMan probes, Molecular Beacons can be expensive to synthesize, with a separate probe required for each target.

Scorpions

With Scorpion probes, sequence-specific priming and PCR product detection is achieved using a single oligonucleotide. The Scorpion probe maintains a stem-loop configuration in the unhybridized state. The fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end. The 3' portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 5' end of a specific primer via a non-amplifiable monomer. After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence from being quenched and a signal is observed.

SYBR Green

SYBR Green provides the simplest and most economical format for detecting and quantitating PCR products in real-time reactions. SYBR Green binds double-stranded DNA, and upon excitation emits light. Thus, as a PCR product accumulates, fluorescence increases. The advantages of SYBR Green are that it is inexpensive, easy to use, and sensitive. The disadvantage is that SYBR Green will bind to any double-stranded DNA in the reaction, including primer-dimers and other non-specific reaction products, which results in an overestimation of the target concentration. For single PCR product reactions with well designed primers, SYBR Green can work extremely well, with spurious non-specific background only showing up in very late cycles.

SYBR Green is the most economical choice for real-time PCR product detection. Since the dye binds to double-stranded DNA, there is no need to design a probe for any particular target being analyzed. However, detection by SYBR Green requires extensive optimization. Since the dye cannot distinguish between specific and non-specific product accumulated during PCR, follow up assays are needed to validate results.

Real-time Reporters for Multiplex PCR

TaqMan probes, Molecular Beacons and Scorpions allow multiple DNA species to be measured in the same sample (multiplex PCR), since fluorescent dyes with different emission spectra may be attached to the different probes. Multiplex PCR allows internal controls to be co-amplified and permits allele discrimination in single-tube, homogeneous assays. These hybridization probes afford a level of discrimination impossible to obtain with SYBR Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other spurious products.

Quantitation of Results

Two strategies are commonly employed to quantify the results obtained by real-time RT-PCR; the standard curve method and the comparative threshold method. These are discussed briefly below.

Standard Curve Method

In this method, a standard curve is first constructed from an RNA of known concentration. This curve is then used as a reference standard for extrapolating quantitative information for mRNA targets of unknown concentrations. Though RNA standards can be used, their stability can be a source of variability in the final analyses. In addition, using RNA standards would involve the construction of cDNA plasmids that have to be in vitro transcribed into the RNA standards and accurately quantitated, a time-consuming process. However, the use of absolutely quantitated RNA standards will help generate absolute copy number data.

In addition to RNA, other nucleic acid samples can be used to construct the standard curve, including purified plasmid dsDNA, in vitro generated ssDNA or any cDNA sample expressing the target gene. Spectrophotometric measurements at 260 nm can be used to assess the concentration of these DNAs, which can then be converted to a copy number value based on the molecular weight of the sample used. cDNA plasmids are the preferred standards for standard curve quantitation. However, since cDNA plasmids will not control for variations in the efficiency of the reverse transcription step, this method will only yield information on relative changes in mRNA expression. This, and variation introduced due to variable RNA inputs, can be corrected by normalization to a housekeeping gene.

Comparative Ct Method

Another quantitation approach is termed the comparative Ct method. This involves comparing the Ct values of the samples of interest with a control or calibrator such as a non-treated sample or RNA from normal tissue. The Ct values of both the calibrator and the samples of interest are normalized to an appropriate endogenous housekeeping gene.

The comparative Ct method is also known as the 2–[delta][delta]Ct method, where

[delta][delta]Ct = [delta]Ct,sample - [delta]Ct,reference

Here, [delta]CT,sample is the Ct value for any sample normalized to the endogenous housekeeping gene and [delta]Ct, reference is the Ct value for the calibrator also normalized to the endogenous housekeeping gene.

For the [delta][delta]Ct calculation to be valid, the amplification efficiencies of the target and the endogenous reference must be approximately equal. This can be established by looking at how [delta]Ct varies with template dilution. If the plot of cDNA dilution versus delta Ct is close to zero, it implies that the efficiences of the target and housekeeping genes are very similar. If a housekeeping gene cannot be found whose amplification efficiency is similar to the target, then the standard curve method is preferred.

Instrumentation for Real-Time PCR

Real-time PCR requires an instrumentation platform that consists of a thermal cycler, a computer, optics for fluorescence excitation and emission collection, and data acquisition and analysis software. These machines, available from several manufacturers, differ in sample capacity (some are 96-well standard format, others process fewer samples or require specialized glass capillary tubes), method of excitation (some use lasers, others broad spectrum light sources with tunable filters), and overall sensitivity. There are also platform-specific differences in how the software processes data. Real-time PCR machines are not inexpensive, currently about $25K - $95K, but are well within purchasing reach of core facilities or labs that have the need for high throughput quantitative analysis. For a comprehensive list of real-time thermal cyclers please see the weblink at the end of this article.

Tools for Real-Time RT-PCR

Ambion’s MessageSensor™ RT Kit includes an RNase H+ MMLV RT that clearly outperforms MMLV RT enzymes that have abolished RNase H activity in real-time RT-PCR experiments. Unlike many other qRT-PCR kits, MessageSensor includes a total RNA control, a control human GAPDH primer set, RNase inhibitor, and nucleotides, as well as a buffer additive that enables detection with SYBR® Green dye.

The Cells-to-cDNA™ II Kit produces cDNA from cultured mammalian cells in less than 2 hours. No RNA isolation is required. This kit is ideal for those who want to perform reverse transcription reactions on small numbers of cells, numerous cell samples, or for scientists who are unfamiliar with RNA isolation. Ambion's Cells-to-cDNA II Kit contains a novel Cell Lysis Buffer that inactivates endogenous RNases without compromising downstream enzymatic reactions. After inactivation of RNases, the cell lysate can be directly added to a cDNA synthesis reaction. Cells-to-cDNA II is compatible with both one-step and two-step real-time RT-PCR protocols.

Genomic DNA contamination can lead to false positive RT-PCR results. Ambion offers a variety of tools for eliminating genomic DNA contamination from RNA samples prior to RT-PCR. Ambion’s DNA-free™ DNase Treatment and Removal Reagents are designed for removing contaminating DNA from RNA samples and for the removal of DNase after treatment without Proteinase K treatment and organic extraction. In addition, Ambion has also developed TURBO™ DNase, a hyperactive enzyme engineered from wild-type bovine DNase. The proficiency of TURBO DNase in binding very low concentrations of DNA means that the enzyme is particularly effective in removing trace quantities of DNA contamination.

Ambion now also offers an economical alternative to the high cost of PCR reagents for the ABI 7700 and other 0.2 ml tube-based real-time instruments. SuperTaq™ Real-Time performs as well or better than the more expensive alternatives, and includes dNTPs and a Reaction Buffer optimized for SYBR Green, TaqMan, and Molecular Beacon chemistries.

End-Point RT-PCR: Relative vs. Competitive vs. Comparative

In spite of the rapid advances made in the area of real-time PCR detection chemistries and instrumentation, end-point RT-PCR still remains a very commonly used technique for measuring changes in gene-expression in small sample numbers.

End-point RT-PCR can be used to measure changes in expression levels using three different methods: relative, competitive and comparative. The most commonly used procedures for quantitating end-point RT-PCR results rely on detecting a fluorescent dye such as ethidium bromide, or quantitation of P32-labeled PCR product by a phosphorimager or, to a lesser extent, by scintillation counting.

Relative quantitation compares transcript abundance across multiple samples, using a co-amplified internal control for sample normalization. Results are expressed as ratios of the gene-specific signal to the internal control signal. This yields a corrected relative value for the gene-specific product in each sample. These values may be compared between samples for an estimate of the relative expression of target RNA in the samples; for example, 2.5-fold more IL-12 in sample 2 than in sample 1.

Absolute quantitation, using competitive RT-PCR, measures the absolute amount (e.g., 5.3 x 105 copies) of a specific mRNA sequence in a sample. Dilutions of a synthetic RNA (identical in sequence, but slightly shorter than the endogenous target) are added to sample RNA replicates and are co-amplified with the endogenous target. The PCR product from the endogenous transcript is then compared to the concentration curve created by the synthetic "competitor RNA."

Comparative RT-PCR mimics competitive RT-PCR in that target message from each RNA sample competes for amplification reagents within a single reaction, making the technique reliably quantitative. Because the cDNA from both samples have the same PCR primer binding site, one sample acts as a competitor for the other, making it unnecessary to synthesize a competitor RNA sequence.

Both relative and competitive RT-PCR quantitation techniques require pilot experiments. In the case of relative RT-PCR, pilot experiments include selection of a quantitation method and determination of the exponential range of amplification for each mRNA under study. For competitive RT-PCR, a synthetic RNA competitor transcript must be synthesized and used in pilot experiments to determine the appropriate range for the standard curve. Comparative RT-PCR yields similar sensitivity as relative and competitive RT-PCR, but requires significantly less optimization and does not require synthesis of a competitor.

Relative RT-PCR

Relative RT-PCR uses primers for an internal control that are multiplexed in the same RT-PCR reaction with the gene specific primers. Internal control and gene-specific primers must be compatible — that is, they must not produce additional bands or hybridize to each other. The expression of the internal control should be constant across all samples being analyzed. Then the signal from the internal control can be used to normalize sample data to account for tube-to-tube differences caused by variable RNA quality or RT efficiency, inaccurate quantitation or pipetting. Common internal controls include ß-actin and GAPDH mRNAs and 18S rRNA. Unlike Northerns and nuclease protection assays, where an internal control probe is simply added to the experiment, the use of internal controls in relative RT-PCR requires substantial optimization.

For relative RT-PCR data to be meaningful, the PCR reaction must be terminated when the products from both the internal control and the gene of interest are detectable and are being amplified within exponential phase (see Determining Exponential Range in PCR). Because internal control RNAs are typically constituitively expressed housekeeping genes of high abundance, their amplification surpasses exponential phase with very few PCR cycles. It is therefore difficult to identify compatible exponential phase conditions where the PCR product from a rare message is detectable. Detection methods with low sensitivity, like ethidium bromide staining of agarose gels, are therefore not recommended. Detecting a rare message while staying in exponential range with an abundant message can be achieved several ways: 1) by increasing the sensitivity of product detection, 2) by decreasing the amount of input template in the RT or PCR reactions and/or 3) by decreasing the number of PCR cycles.

Ambion recommends using 18S rRNA as an internal control because it shows less variance in expression across treatment conditions than ß-actin and GAPDH. However, because of its abundance, it is difficult to detect the PCR product for rare messages in the exponential phase of amplification of 18S rRNA. Ambion's patented Competimer™ Technology solves this problem by attenuating the 18S rRNA signal even to the level of rare messages. Attenuation results from the use of competimers — primers identical in sequence to the functional 18S rRNA primers but that are "blocked" at their 3'-end and, thus, cannot be extended by PCR. Competimers and primers are mixed at various ratios to reduce the amount of PCR product generated from 18S rRNA. Figure 1 illustrates that 18S rRNA primers without competimers cannot be used as an internal control because the 18S rRNA amplification overwhelms that of clathrin (compare panels A and B). Mixing primers with competimers at a 3:7 ratio attenuates the 18S rRNA signal, making 18S rRNA a practical internal control (panel C).

Ambion's QuantumRNA 18S Internal Standards contain 18S rRNA primers and competimers designed to amplify 18S rRNA in all eukaryotes. The Universal 18S Internal Standards function across the broadest range of organisms including plants, animals and many protozoa. The Classic I and Classic II 18S Internal Standards can be used with any vertebrate RNA sample. All 18S Internal Standards work well in multiplex RT-PCR. These kits also include control RNA and an Instruction Manual detailing the series of experiments needed to make relative RT-PCR data significant. For those researchers who have validated ß-actin as an appropriate internal control for their system, the QuantumRNA ß-actin Internal Standards are available.

Competitive RT-PCR

Competitive RT-PCR precisely quantitates a message by comparing RT-PCR product signal intensity to a concentration curve generated by a synthetic competitor RNA sequence. The competitor RNA transcript is designed for amplification by the same primers and with the same efficiency as the endogenous target. The competitor produces a different-sized product so that it can be distinguished from the endogenous target product by gel analysis. The competitor is carefully quantitated and titrated into replicate RNA samples. Pilot experiments are used to find the range of competitor concentration where the experimental signal is most similar. Finally, the mass of product in the experimental samples is compared to the curve to determine the amount of a specific RNA present in the sample.

Some protocols use DNA competitors or random sequences for competitive RT-PCR. These competitors do not effectively control for variations in the RT reaction or for the amplification efficiency of the specific experimental sequence, as do RNA competitors. See The Accuracy of Competitive RT-PCR Depends on Using the Right Exogenous Standard for a further discussion on competitor choice and design.

Comparative RT-PCR

While exquisitely sensitive, both relative and competitive methods of qRT-PCR have drawbacks. Relative RT-PCR requires extensive optimization to ensure that the PCR is terminated when both the gene of interest and an internal control are in the exponential phase of amplification. Competitive RT-PCR requires that an exogenous "competitor" be synthesized for each target to be analyzed. However, comparative RT-PCR achieves the same level of sensitivity as these standard methods of qRT-PCR, with significantly less optimization. Target mRNAs from 2 samples are assayed simultaneously, each serving as a competitor for the other, making it possible to compare the relative abundance of target between samples. Comparative RT-PCR is ideal for analyzing target genes discovered by screening methods such as array analysis and differential display.

Tools for Any RT-PCR Technique

Whether you choose to perform real-time, relative, competitive, or comparative RT-PCR, Ambion offers products to simplify your RT-PCR experiments and make the data more quantitative. In addition to the specific products described above, Ambion offers SuperTaq™ Polymerase, M-MLV Reverse Transcriptase, and RNase-free PCR tubes. To prevent cross contamination during PCR experiments, Ambion also offers DNAZap™ DNA Degradation Solution and RNase-free barrier pipette tips.

For a comprehensive list of publications discussing practically every aspect of real-time RT-PCR please visit www.wzw.tum.de/gene-quantification/real-time.html

Ten Most Common Real-Time qRT-PCR Pitfalls

2007-12-18T14:18:00.000-05:00

Ten Most Common Real-Time qRT-PCR Pitfalls

1- Poor primer and probe design. For the most efficient design of PCR primer and probe sets for real-time qRT-PCR, we strongly recommend using primer design software. Most primer design programs include adjustable parameters for optimal primer and probe design. These parameters consider primer/probe Tm, complementarity, and secondary structure as well as amplicon size and other important factors. Restricting the number of identical nucleotide runs is also recommended. When designing amplicons in eukaryotic targets, choose PCR primers that span at least one exon-exon junction in the target mRNA to prevent amplification of the target from contaminating genomic DNA.

2- Using poor quality RNA. Degraded or impure RNA can limit the efficiency of the RT reaction and reduce yield. RNA should either be prepared from fresh tissue, or from tissue treated with an RNA stabilization solution such as RNAlater® (see www.ambion.com/techlib/
resources/rnalater for more information). The importance of using full length RNA for reverse transcription depends on the application. Amplicons for real-time qRT-PCR are typically short (70-250 bp). As a result, some degradation of the RNA can be tolerated. If it is not possible to use completely intact RNA, design primers to anneal to an internal region of the gene of interest. Note that for truly quantitative RT-PCR, partially degraded RNA may not give an accurate representation of gene expression.

3- Not using "master mixes". qRT-PCR is a highly sensitive tool for analyzing RNA. As the PCR amplifies the target, errors are simultaneously amplified. Therefore, variability should be kept to a minimum whenever possible. A "master mix", or mixture of the reaction reagents, should be used when setting up multiple reactions to minimize sample-to-sample and well-to-well variation and improve reproducibility. To further reduce well-to-well variation, a reference dye such as ROX can be added to the master mix.

4- Introducing cross-contamination. All surfaces in the PCR area should be routinely decontaminated to prevent cross contamination use of a DNA decontamination solution, such as DNAzap™, that destroys DNA, is recommended. A "No Template Control" (NTC) should be run to rule out cross contamination of reagents and surfaces. The NTC includes all of the RT-PCR reagents except the RNA template. Typically the RNA is simply substituted with nuclease-free water. No product should be synthesized in the NTC; if a product is amplified, it indicates that one or more of the RT-PCR reagents is contaminated with the amplicon.

5- Not using a "– RT" control. It is virtually impossible to completely eliminate genomic DNA from RNA preparations. Therefore, it is important to include a minus-reverse transcriptase control ("No Amplification Control" or NAC) in qRT-PCR experiments. Typically, the NAC is a mock reverse transcription containing all the RT-PCR reagents, except the reverse transcriptase. If a product is seen in the NAC, it probably indicates that contaminating DNA is present in the sample.

6- Using an inappropriate normalization control. The reliability of any qRT-PCR experiment can be improved by including an invariant endogenous control in the assay to correct for sample to sample variations in qRT-PCR efficiency and errors in sample quantitation. The expression level of a good control should not vary across the samples being analyzed. 18S rRNA is often used as a control because it is less variant in expression level than other traditional internal controls such as ß-actin or GAPDH. For more information read Using 18S rRNA as an Internal Control for Relative RT-PCR.

7- Dissociation (melting) curves are not performed when using SYBR® Green. Ideally, the experimental samples should yield a sharp peak (first derivative plot) at the melting temperature of the amplicon, whereas the NAC and NTC will not generate significant fluorescent signal. This result indicates that the products are specific, and that SYBR Green I fluorescence is a direct measure of accumulation of the product of interest. If the dissociation curve reveals a series of peaks, it indicates that there is not enough discrimination between specific and non-specific reaction products. To obtain meaningful data, optimization of the qRT-PCR would be necessary.

8- Not setting the baseline and threshold properly. To obtain accurate Ct values the baseline needs to be set two cycles earlier than the Ct value for the most abundant sample. For real-time qRT-PCR data to be meaningful, the threshold should be set when the product is in exponential phase. Typically this is set at least 10 standard deviations from of the baseline.

9-The efficiency of the reaction is poor. The efficiency (Eff) of the reaction can be calculated by the following equation:

Eff=10^(-1/slope) –1

The efficiency of the PCR should be 90-110% (3.6 > slope > 3.1), A number of variables can affect the efficiency of the PCR. These factors can include length of the amplicon, secondary structure, and primer design, to name a few. Although valid data can be obtained that fall outside of the efficiency range, the qRT-PCR should be further optimized or alternative amplicons designed.

10- Using an inappropriate range for standard curves. Standard curves should be prepared for each gene under study for RNA quantitation (absolute or relative quantitation), or for verification of the efficiencies of the reactions for comparative quantitation (delta-delta-Ct). The standard curve should extend above and below the expected abundance of your target. Additional input quantities can be included such as the minimum and maximum RNA amounts above and below the limit of detection to help differentiate between specific and non-specific products.

Antibody Books!

2007-12-05T11:20:00.000-05:00

Introduction to Antibodies (Chemicon)(PDF 2.38 MB) - Chemicon International Inc. is pleased to offer the second edition of Introduction to Antibodies, a basic guide to immunological assays and general technical information. This is a handy reference to supplement those techniques described in the literature, recorded in general laboratory procedures, and described on individual product data sheets. As every antibody and experimental design is unique, these general assay suggestions should not be interpreted as applicable to all situations, but rather as an additional source of reference information describing techniques that have been successfully used for a variety of the antibodies available today. As always, individual assays must be optimized empirically and antibody titers must be established for every unique batch of antibody.

Antibodies Protocol Guide (Clontech) (PDF 67 KB) - CLONTECH’s Antibodies Protocol Guide provides experimental procedures for commonly used antibody techniques. These protocols are intended as starting points for establishing optimal conditions for your experimental system. In some instances, specific, optimized antibody protocols are required. When this is the case, we supply the preferred protocols with the antibody. These optimized protocols should be used instead of the more generalized procedures in this guide.

Monoclonal Antibody Production (Committee on Methods of Producing Monoclonal Antibodies, Institute for Laboratory Animal Research, National Research Council) (PDF 4.63 MB) - Monoclonal antibodies (mAb) are used extensively in basic biomedical research, in diagnosis of disease, and in treatment of illnesses, such as infections and cancer. Antibodies are important tools used by many investigators in their research and have led to many medical advances. Producing mAb requires immunizing an animal, usually a mouse; obtaining immune cells from its spleen; and fusing the cells with a cancer cell (such as cells from a myeloma) to make them immortal, which means that they will grow and divide indefinitely. A tumor of the fused cells is called a hybridoma, and these cells secrete mAb. The development of the immortal hybridoma requires the use of animals; no commonly accepted nonanimal alternatives are available.

Western Blotting Handbook and Troubleshooting Guide (PIERCE) (PDF 2.33 MB) - The Western Blotting Handbook and Troubleshooting Guide is a 60-page booklet that details each step of the Western blotting process with technical information and products for transfer, blocking, washing, antibodies, substrates, film and stripping buffer. You will want to keep this new booklet close at hand because it also includes protocols, references and a troubleshooting guide.

Western Blot Method (LI-COR Biosciences) (PDF 362 KB) - Detailed protocol for Western Blot detection in the Odyssey Imaging System. Published January, 2006. The most recent version of this protocol is posted at http://biosupport.licor.com/support.

The Human IgG Subclasses (Calbiochem) (PDF 620 KB) - We are pleased to present you with this new edition of The Human IgG Subclasses Booklet. As part of our continuing commitment to provide useful product information and exceptional service to our customers, we have compiled this practical resource for investigators who are interested in the rapidly expanding field of quantitation of human immunoglobulins, especially the IgG subclass proteins. Whether you are just beginning your research or are training new researchers in your laboratory, you will find this booklet to be a highly useful reference source.

Buffers: A guide for the preparation and use of buffers in biological systems (Calbiochem)(PDF 320 KB) - We are pleased to present to you the newest edition of Buffers: A Guide for the Preparation and Use of Buffers in Biological Systems. This practical resource has been especially revamped for use by researchers in the biological sciences. This publication is a part of our continuing commitment to provide useful product information and exceptional service to you, our customers. You will find this booklet a highly useful resource, whether you are just beginning your research work or training the newest researchers in your laboratory.

A Guide to the Properties and Uses of Detergents in Biology and Biochemistry (Calbiochem) (PDF 620 KB) - CALBIOCHEM® has been the leading supplier of a variety of quality detergents to a large number of researchers all over the world for almost 50 years. During this period, we have received a number of inquiries on the use of detergents, definitions, relevance of critical micelle concentration (CMC), cloud point, hydrophilic number, and how to select the most appropriate detergent. As a service to the research community, CALBIOCHEM® is providing this guide to the use of detergents in biological systems. The background information and the selected bibliography provided here will hopefully serve the needs of the first time users of detergents as well as those of experienced investigators.

Quick Guide for PCR Vol. I

2007-12-05T11:14:00.000-05:00

The COMPLETE Alkami Quick GuideTM for PCR is a
158 page manual with a file size of 296 KB. It is formated
to be 5.5" X 8.5" in order to make the Guide a convenient
size for use at your bench. The complete Guide will print
out on 79 single-sided, letter sized, 8.5" X 11" sheets
which you can cut and bind as desired.

REAL-TIME PCR

2007-12-05T11:08:00.000-05:00

Dorak MT (Ed): Real-Time PCR (Advanced Methods Series). Oxford: Taylor & Francis, 2006
(Amazon) (Table of Contents)

Glossary of Terms Used in Real-Time PCR

PowerPoint Presentation on Real-Time PCR

Real-time reverse-transcriptase (RT) PCR quantitates the initial amount of the template most specifically, sensitively and reproducibly, and is a preferable alternative to other forms of quantitative RT-PCR that detect the amount of final amplified product at the end-point 1 2 (Freeman, 1999; Raeymaekers, 2000). Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle (ie, in real time) as opposed to the endpoint detection 3,4 (Higuchi, 1992; Higuchi, 1993). The real-time progress of the reaction can be viewed in some systems. Real-time PCR does not detect the size of the amplicon and thus does not allow the differentiation between DNA and cDNA amplification, however, it is not influenced by non-specific amplification unless SYBR Green is used (see below). Real-time PCR quantitation (qPCR) eliminates post-PCR processing of PCR products (which is necessary in competitive RT-PCR). This helps to increase throughput and reduce the chances of carryover contamination. In comparison to conventional RT-PCR, real-time PCR also offers a much wider dynamic range of up to 107-fold (compared to 1000-fold in conventional RT-PCR). Dynamic range of any assay determines how much target concentration can vary and still be quantified. A wide dynamic range means that a wide range of ratios of target and normalizer can be assayed with equal sensitivity and specificity. It follows that the broader the dynamic range, the more accurate the quantitation.

The real-time PCR system is based on the detection and quantitation of a fluorescent reporter 5,6 (Lee, 1993; Livak, 1995). This signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. A significant increase in fluorescence above the baseline value measured during the 3-15 cycles indicates the detection of accumulated PCR product.

A fixed fluorescence threshold is set significantly above the baseline that can be altered by the operator. The parameter CT (threshold cycle) is defined as the cycle number at which the fluorescence emission exceeds the fixed threshold. There are three main fluorescence-monitoring systems for DNA amplification 7 (Wittwer, 1997a): (1) hydrolysis probes; (2) hybridizing probes (see Hybridization Probe Chemistry); and (3) DNA-binding agents 8,9 (Wittwer, 1997b; van der Velden, 2003). Hydrolysis probes include TaqMan probes 10 (Heid, 1996), molecular beacons 11-15 (Mhlanga, 2001; Vet, 2002; Abravaya, 2003; Tan, 2004; Vet & Marras, 2005) and scorpions (further details) 16-18 (Saha, 2001; Solinas, 2001; Terry, 2002). They use the fluorogenic 5' exonuclease activity of Taq polymerase to measure the amount of target sequences in cDNA samples (see also 19 Svanvik, 2000 for light-up probes).

TaqMan probes are oligonucleotides longer than the primers (20-30 bases long with a Tm value of 10 oC higher) that contain a fluorescent dye usually on the 5' base, and a quenching dye (usually TAMRA) typically on the 3' base (TaqMan MGB probes have a non-fluorescent quencher and minor groove binder at the 3’ end). When irradiated, the excited fluorescent dye transfers energy to the nearby quenching dye molecule rather than fluorescing (this is called FRET = Förster or fluorescence resonance energy transfer) 20,21 (Hiyoshi, 1994; Chen, 1997). Thus, the close proximity of the reporter and quencher prevents emission of any fluorescence while the probe is intact. TaqMan probes are designed to anneal to an internal region of a PCR product. When the polymerase replicates a template on which a TaqMan probe is bound, its 5' exonuclease activity cleaves the 5’ end of probe which contains the reporter dye 22 (Holland, 1991). This ends the activity of quencher (no FRET) and the reporter dye starts to emit fluorescence which increases in each cycle proportional to the rate of probe cleavage. Accumulation of PCR products is detected by monitoring the increase in fluorescence of the reporter dye (note that primers are not labeled). TaqMan assay uses universal thermal cycling parameters and PCR reaction conditions. Because the cleavage occurs only if the probe hybridizes to the target, the origin of the detected fluorescence is specific amplification. The process of hybridization and cleavage does not interfere with the exponential accumulation of the product. One specific requirement for fluorogenic probes is that there be no G at the 5' end. A 'G' adjacent to the reporter dye quenches reporter fluorescence even after cleavage. Well-designed TaqMan probes require very little optimization (see a list of SNP500 Cancer Validated TaqMan Allelic Discrimination Assays).

Molecular beacons also contain fluorescent (FAM, TAMRA, TET, ROX) and quenching dyes (typically DABCYL) at either end but they are designed to adopt a hairpin structure while free in solution to bring the fluorescent dye and the quencher in close proximity for FRET to occur. They have two arms with complementary sequences that form a very stable hybrid or stem. The close proximity of the reporter and the quencher in this hairpin configuration suppresses reporter fluorescence. When the beacon hybridizes to the target during the annealing step, the reporter dye is separated from the quencher and the reporter fluoresces (FRET does not occur). Molecular beacons remain intact during PCR and must rebind to target every cycle for fluorescence emission. This will correlate to the amount of PCR product available. All real-time PCR chemistries allow detection of multiple DNA species (multiplexing) by designing each probe/beacon with a spectrally unique fluor/quench pair, or if SYBR green is used by melting curve analysis. By multiplexing, the target(s) and endogenous control can be amplified in single tube for qPCR purposes. For examples, see 23-31 (Bernard, 1998; Vet, 1999; Lee, 1999; Donohoe, 2000; Read, 2001; Grace, 2003; Vrettou, 2004; Rickert, 2004; Persson, 2005.

With Scorpion primer/probes, sequence-specific priming and PCR product detection is achieved using a single oligonucleotide. The Scorpion probe maintains a stem-loop configuration in the unhybridized state. The fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end. The 3' portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 5' end of a specific primer via a non-amplifiable monomer. After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence from being quenched and a signal is observed (see also How It Works)

The cheaper alternative is the double-stranded DNA binding dye chemistry, which quantitates the amplicon production (including non-specific amplification and primer-dimer complex) by the use of a non-sequence specific fluorescent intercalating agent (SYBR-green I or ethidium bromide). It does not bind to ssDNA. SYBR green is a fluorogenic minor groove binding dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal upon binding to double-stranded DNA 32 (Morrison, 1998). Disadvantages of SYBR green-based real-time PCR include the requirement for extensive optimization. Furthermore, non-specific amplifications require follow-up assays (melting point or dissociation curve analysis) for amplicon identification 33 (Ririe, 1997). The method has been used in HFE-C282Y genotyping 26 (Donohoe, 2000). Another controllable problem is that longer amplicons create a stronger signal (if combined with other factors, this may cause CDC camera saturation, see below). Normally SYBR green is used in singleplex reactions, however when coupled with melting curve analysis, it can be used for multiplex reactions 34 (Siraj, 2002).

The threshold cycle or the CT value is the cycle at which a significant increase in DRn is first detected (for definition of DRn, see below and glossary). The threshold cycle is when the system begins to detect the increase in the fluorescent signal associated with an exponential growth of PCR product during the log-linear phase. This phase provides the most useful information about the reaction (certainly more important than the end-point). The slope of the log-linear phase reflects the amplification efficiency (Eff). Eff can be calculated by the formula:

Eff = 10(-1/slope) – 1

The efficiency of the PCR should be 90 - 100% (– 3.6 > slope > – 3.1) (Stratagene Slope to Efficiency Calculator). A number of variables can affect the efficiency of the PCR 35-37 (Bustin, 2004; Wong, 2005; Yuan, 2006). These factors include length of the amplicon, secondary structure and primer quality. Although valid data can be obtained that fall outside of the efficiency range, the qRT-PCR should be further optimized or alternative amplicons designed (see Efficiency Determination Page by Pfaffl). For the slope to be an indicator of real amplification (rather than signal drift), there has to be an inflection point. This is the point on the growth curve when the log-linear phase begins. It also represents the greatest rate of change along the growth curve. (Signal drift is characterized by gradual increase or decrease in fluorescence without amplification of the product.) The important parameter for quantitation is the CT. The higher the initial amount of genomic DNA, the sooner accumulated product is detected in the PCR process, and the lower the CT value. The threshold should be placed above any baseline activity and within the exponential increase phase (which looks linear in the log transformation). Some software allows determination of the cycle threshold (CT) by a mathematical analysis of the growth curve. This provides better run-to-run reproducibility. A CT value of 40 or higher means no amplification and this value cannot be included in the calculations. Besides being used for quantitation, the CT value can be used for qualitative analysis as a pass/fail measure.

Relative gene expression comparisons work best when the gene expression of the chosen endogenous/internal control is more abundant and remains constant, in proportion to total RNA, among the samples. By using an invariant endogenous control as an active reference, quantitation of an mRNA target can be normalized for differences in the amount of total RNA added to each reaction. For this purpose, the most common choices are 18S RNA, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and b-actin. Because the 18S mRNA does not have a poly-A tail, cDNA synthesis using oligo-dT should not be used if 18S RNA will be used as a normalizer. The issue of the choice of a normalizer has been reviewed by Suzuki et al. 38 (Suzuki, 2000). The authors recommend caution in the use of GAPDH as a normalizer as it has been shown that its expression may be upregulated in proliferating cells. They recommend b-actin as a better active reference. GAPDH is severely criticized as a normalizer by others too 39-41 (Bustin SA, 2000; Dheda, 2004; Aerts, 2004). GAPDH is particularly an unpopular choice in cancers because of its increased expression in aggressive cancers 42 (Goidin, 2001). Caution should also be exercised when 18S RNA is used as a normalizer as it is a ribosomal RNA species (not mRNA) and may not always represent the overall cellular mRNA population. Since the chosen mRNA species should be proportional to the amount of input RNA, it may be best to use a combination as normalizer. It is desirable to validate the chosen normalizer for the target cell or tissue. It should be expressed at a constant level at different time points by the same individual and also by different individuals at the target cell or tissue (for example, peripheral blood lymphocytes) 40 (Dheda, 2004). This aim can be achieved by the ABI's TaqMan Human Endogenous Control Plate or TATAA Biocenter's Endogenous Control Gene Panel which evaluate the expression of select housekeeping genes. Our own experience 43 (Sabek, 2002) showed that b-actin or 18S RNA are the best choices as normalizers for the peripheral blood mononuclear cells, whereas GAPDH performed worst in transplant monitoring studies. Similar concerns on the choice of normalizers in transplant monitoring have also been expressed by others 44 (Gibbs, 2003). Surveys of tumor cell lines or tissues reported the worst results with GAPDH while beta-glucuronidase (GUS) and 18S rRNA were the best choices for this target 41 (Aerts, 2004) or HPRT 45 (de Kok, 2004). It is important to choose a normalizer whose expression will remain constant under the experimental conditions designed for the target gene 46 (Schmittgen, 2000). Another study found that for robust conclusions the use of multiple internal controls is the best choice (18S rRNA and cyclophilin) for kidney mRNA expression studies 47 (Schmid, 2003). The strategy of using multiple and variable normalizer genes depending on the cell and tissue type is validated for general use 48 (Vandesompele, 2002). (See Ambion: 18S RNA as an Internal Control; Ambion: GAPDH, b-actin, cyclophilin, 18S RNA as internal controls)

Multiplex TaqMan assays can be performed using multiple dyes with distinct emission wavelengths. Available dyes for this purpose are FAM, TET, VIC and JOE (the most expensive). TAMRA is reserved as the quencher on the traditional TaqMan probes and ROX as the passive reference. For best results, the combination of FAM (target) and VIC (endogenous control) is recommended (they have the largest difference in emission maximum) whereas JOE and VIC should not be combined. It is important that if the dye layer has not been chosen correctly, the machine will still read the other dye's spectrum. For example, both VIC and FAM emit fluorescence in a similar range to each other and when doing a single dye, the wells should be labeled correctly. In the case of multiplexing, the spectral compensation for the post run analysis should be turned on (on ABI 7700: Instrument/Diagnostics/Advanced Options/Miscellaneous). Activating spectral compensation improves dye spectral resolution.

One-step real-time RT-PCR performs reverse transcription and PCR in a single buffer system and in one tube. In two-step RT-PCR, these two steps are performed separately in different tubes.

TaqMan primer and probe design guidelines
1. The Primer Express software designs primers with a melting temperature (Tm) of 58-600 C, and probes with a Tm value of 100 C higher. The Tm of both primers should be equal,
2. Primers should be 15-30 bases in length (protocol),
3. The G+C content should ideally be 30-80%. If a higher G+C content is unavoidable, the use of high annealing and melting temperatures, cosolvents such as glycerol, DMSO, or 7-deaza-dGTP may be necessary,
4. The run of an identical nucleotide should be avoided. This is especially true for G, where runs of four or more Gs is not allowed,
5. The total number of Gs and Cs in the last five nucleotides at the 3' end of the primer should not exceed two (the newer version of the software has an option to do this automatically but not the original version). This helps to introduce relative instability to the 3' end of primers to reduce non-specific priming. The primer conditions are the same for SYBR Green assays,
6. Maximum amplicon size should not exceed 400 bp (ideally 50-150 bases). Smaller amplicons give more consistent results because PCR is more efficient and more tolerant of reaction conditions (the short length requirement has nothing to do with the efficiency of 5' nuclease activity),
7. The probes should not have runs of identical nucleotides (especially four or more consecutive Gs), G+C content should be 30-80%, there should be more Cs than Gs, and not a G at the 5' end. The higher number of Cs produces a higher DRn (this feature may require manual check). The choice of probe should be made first,
8. To avoid false-positive results due to amplification of contaminating genomic DNA in the cDNA preparation, it is preferable to have primers spanning exon-exon junctions in the cDNA sequence. This way, genomic DNA will not be amplified,
9. If a TaqMan probe is designed for allelic discrimination, the mismatching nucleotide (the polymorphic site) should be in the middle of the probe rather than at the ends,
10. Use primers that contain dA nucleotides near the 3' ends so that any primer-dimer generated is efficiently degraded by AmpErase UNG (mentioned in p.9 of the manual for EZ RT-PCR kit; P/N 402877). If primers cannot be selected with dA nucleotides near the ends, the use of primers with 3' terminal dU-nucleotides should be considered.
See also ABgene Dual Labeled Probe Design Guide.

General recommendations for real-time RT-PCR
1. Use positive-displacement pipettes to avoid inaccuracies in pipeting,
2. The sensitivity of real-time PCR allows detection of the target in 2 pg of total RNA. The number of copies of total RNA used in the reaction should ideally be enough to give a signal by 25-30 cycles (preferably less than 100 ng). The amount used should be decreased or increased to achieve this,
3. The optimal concentrations of the reagents are as follows:
i. Magnesium chloride concentration should be between 4 and 7 mM. It is optimized as 5.5 mM for the primers/probes designed using the Primer Express software,
ii. Concentrations of dNTPs should be balanced with the exception of dUTP (if used). Substitution of dUTP for dTTP for control of PCR product carryover requires twice dUTP that of other dNTPs. While the optimal range for dNTPs is 500 mM to 1 mM (for one-step RT-PCR), for a typical TaqMan reaction (PCR only), 200 mM of each dNTP (400 mM of dUTP) is used,
iii. Typically 0.25 mL (1.25 U) AmpliTaq DNA Polymerase (5.0 U/mL) is added into each 50 mL reaction. This is the minimum requirement. If necessary, optimization can be done by increasing this amount by 0.25 U increments,
iv. The optimal probe concentration is 50-200 nM, and the primer concentration is 100-900 nM. Ideally, each primer pair should be optimized at three different temperatures (58, 60 and 620 C for TaqMan primers) and at each combination of three concentrations (50, 300, 900 nM). This means setting up three different sets (for three temperatures) with nine reactions in each (50/50 mM, 50/300 mM, 50/900, 300/50, 300/300, 300/900, 900/50, 900/300, 900/900 mM) using a fixed amount of target template. If necessary, a second round of optimization may improve the results. Optimal performance is achieved by selecting the primer concentrations that provide the lowest CT and highest DRn. Similarly, the probe concentration should be optimized for 25-225 nM,
4. If AmpliTaq Gold DNA Polymerase is being used, there has to be a 9-12 min pre-PCR heat step at 92 - 950 C to activate it. If AmpliTaq Gold DNA Polymerase is used, there is no need to set up the reaction on ice. A typical TaqMan reaction consists of 2 min at 500 C for UNG (see below) incubation, 10 min at 950 C for Polymerase activation, and 40 cycles of 15 sec at 950 C (denaturation) and 1 min at 600 C (annealing and extension). A typical reverse transcription cycle (for cDNA synthesis), which should precede the TaqMan reaction if the starting material is total RNA, consists of 10 min at 250 C (primer incubation), 30 min at 480 C (reverse transcription with conventional reverse transcriptase) and 5 min at 950 C (reverse transcriptase inactivation),
5. AmpErase uracil-N-glycosylase (UNG) is added in the reaction to prevent the reamplification of carry-over PCR products by removing any uracil incorporated into amplicons. This is why dUTP is used rather than dTTP in PCR reaction. UNG does not function above 55 0C and does not cut single-stranded DNA with terminal dU nucleotides 49 (Longo, 1990). UNG-containing master mix should not be used with one-step RT-PCR unless rTth DNA polymerase is being used for reverse transcription and PCR (TaqMan EZ RT-PCR kit),
6. It is necessary to include at least three No Amplification Controls (NAC, a minus-reverse transcriptase control) as well as three No Template Controls (NTC, a minus sample control) in each reaction plate (to achieve a 99.7% confidence level in the definition of +/- thresholds for the target amplification, six replicates of NTCs must be run). NAC is a mock reverse transcription containing all the RT-PCR reagents, except the reverse transcriptase; NTC includes all of the RT-PCR reagents except the RNA template. It is necessary to rule out the presence of fluorescence contaminants in the sample or in the heat block of the thermal cycler (these would cause false positives). No product should be synthesized in the NTC or NAC; if a product is amplified, it indicates that one or more of the RT-PCR reagents is contaminated with DNA which may be the amplicon. If the absolute fluorescence of the NAC is greater than that of the NTC after PCR, fluorescent contaminants may be present in the sample or in the heating block of the thermal cycler,
7. The dynamic range of a primer/probe system and its normalizer should be examined if the DDCT method is going to be used for relative quantitation. The linear dynamic range refers to the range of initial template concentrations over which accurate CT values are obtained. This is determined by running (in triplicate) reactions of five RNA concentrations (for example, 0, 80 pg/mL, 400 pg/mL, 2 ng/mL and 50 ng/mL). The resulting plot of log of the initial amount vs CT values (standard curve) should be a (near) straight line for both the target and normalizer real-time PCRs for the same range of total RNA concentrations,
8. The passive reference is a dye (ROX) included in the reaction (present in the TaqMan universal PCR master mix). It does not participate in the 5' nuclease reaction. It provides an internal reference for background fluorescence emission. This is used to normalize the reporter-dye signal. This normalization is for non-PCR-related fluorescence fluctuations occurring in different wells (concentration or volume differences, bubbles) or over time and different from the normalization for the amount of cDNA or efficiency of the PCR. Normalization is achieved by dividing the emission intensity of reporter dye by the emission intensity of the passive reference. This gives the ratio defined as Rn. Not using ROX or not designating it as the passive reference dye in the analysis may cause trailing of the clusters in the allelic discrimination plot,
9. In addition to the use of ROX, a master mix should be used when setting up multiple reactions to minimize sample-to-sample and well-to-well variation and improve reproducibility (ROX will be within the master mix),
10. If multiplexing is done, the more abundant of the targets will use up all the ingredients of the reaction before the other target gets a chance to amplify. To avoid this, the primer concentrations for the more abundant target should be limited.
11. If SYBR green is used, dissociation (melting) curve analysis should be performed. Ideally, the experimental samples should yield a sharp peak (first derivative plot) at the melting temperature of the amplicon, whereas the NAC and NTC will not generate significant fluorescent signal. This result indicates that the products are specific, and that SYBR Green I fluorescence is a direct measure of accumulation of the product of interest. If the dissociation curve has a series of peaks, there is not enough discrimination between specific and non-specific reaction products. To obtain meaningful data, optimization of the RT-PCR would be necessary.

Recommendations for the general assay of cDNA samples
1. Reverse transcription of total RNA to cDNA should be done with random hexamers (not with oligo-dT). If oligo-dT has to be used long mRNA transcripts or amplicons greater than two kilobases upstream should be avoided, and 18S RNA cannot be used as normalizer,
2. Multiplex PCR will only work properly if the control primers are limiting (ABI control reagents do not have their primers limited). This requires running primer limiting assays for optimization,
3. The range of target cDNA used is 10 ng to 1 mg. If DNA is used (mainly for allelic discrimination studies), the optimum amount is 100 ng to 1 mg,
4. It is ideal to treat each RNA preparation with RNAse free DNAse to avoid genomic DNA contamination. Even the best RNA extraction methods yield some genomic DNA. Of course, it is ideal to have primers not amplifying genomic DNA at all but sometimes this may not be possible,
5. For optimal results, the reagents (before the preparation of the PCR mix) and the PCR mixture itself (before loading) should be vortexed and mixed well. Otherwise there may be shifting Rn values during the early (0 - 5) cycles of PCR. It is also important to add probe to the buffer component and allow it to equilibrate at room temperature prior to reagent mix formulation.

TaqMan primers and probes
The TaqMan probes ordered from ABI at midi-scale arrive already resuspended at 100 mM. If a 1/20 dilution is made, this gives a 5 mM solution. This stock solution should be aliquoted, frozen and kept in the dark. Using 1 mL of this in a 50 mL reaction gives the recommended 100 nM final concentration.
The primers arrive lyophilized with the amount given on the tube in pmols (such as 150.000 pmol which is equal to 150 nmol). If X nmol of primer is resuspended in X mL of H2O, the resulting solution is 1 mM. It is best to freeze this stock solution in aliquots. When the 1 mM stock solution is diluted 1/100, the resulting working solution will be 10 mM. To get the recommended 50 - 900 nM final primer concentration in 50 mL reaction volume, 0.25 - 4.50 mL should be used per reaction (2.5 mL for 500 nM final concentration). The PDAR primers and probes are supplied as a mix in one tube. They have to be used 2.5 mL in a 50 mL reaction volume.

Setting up one-step TaqMan reaction
One-step real-time PCR uses RNA (as opposed to cDNA) as a template. This is the preferred method if the RNA solution has a low concentration. The disadvantage is that RNA carryover prevention enzyme AmpErase cannot be used in one-step reaction format. In this method, both reverse transcription and real-time PCR take place in the same tube. The downstream PCR primer also acts as the primer for reverse transcriptase (random hexamers or oligo-dT cannot be used for reverse transcription in one-step RT-PCR). One-step reaction requires higher dNTP concentration (³ 300 mM vs 200 mM) as it combines two reactions needing dNTPs in one. A typical reaction mix for one-step PCR by Gold RT-PCR kit is as follows:
H2O + RNA : 20.5 mL (24 mL if PDAR is used)
10X TaqMan buffer : 5.0 mL
MgCl2 (25 mM) : 11.0 mL
dATP (10mM) : 1.5 mL (for final concentration of 300 mM)
dCTP (10mM) : 1.5 mL (for final concentration of 300 mM)
dGTP (10mM) : 1.5 mL (for final concentration of 300 mM)
dUTP (20mM) : 1.5 mL (for final concentration of 600 mM)
Primer F (10 mM) * : 2.5 mL (for final concentration of 500 nM)
Primer R (10 mM) * : 2.5 mL (for final concentration of 500 nM)
TaqMan Probe * : 1.0 mL (for final concentration of 100 nM)
AmpliTaq Gold : 0.25 mL (can be increased for higher efficiency)
Reverse Transcriptase : 0.25 mL
RNAse inhibitor : 1.00 mL
* If a PDAR is used, 2.5 mL of primer + probe mix used.
Ideally 10 pg - 100 ng RNA should be used in this reaction. Note that decreasing the amount of template from 100 ng to 50 ng will increase the CT value by 1. To decrease a CT value by 3, the initial amount of template should be increased 8-fold. ABI claims that 2 picogram RNA can be detected by this system and the maximum amount of RNA that can be used is 1 microgram. For routine analysis, 10 pg - 100 ng RNA and 100 pg - 1 mg genomic DNA can be used.

Cycling parameters for one-step PCR
Reverse transcription (by MuLV) 480 C for 30 min
AmpliTaq activation 950 C for 10 min
PCR: denaturation 950 C for 15 sec and annealing/extension 600 C for 1 min (repeated 40 times) (note that there are only two steps in a real-time PCR cycle)
(On ABI 7700, minimum holding time is 15 seconds.)

The recently introduced EZ one-step RT-PCR kit allows the use of UNG as the incubation time for reverse transcription is 60 0C thanks to the use of a thermostable reverse transcriptase. This temperature is also a better option to avoid primer dimers and non-specific bindings at 48 0C (see also Roche LightCycler One-Step RT-PCR Kit).

Operating ABI 7700
(See also ABI 7000 Compendium)
Make sure the following before starting a run:
1. Cycle parameters are correct for the run (somebody may have used different parameters before you),
2. Choice of spectral compensation is correct (off for singleplex, on for multiplex reactions),
3. Choice of "Number of PCR Stages" is correct in the Analysis Options box (Analysis/Options). This may have to be manually assigned after a run if the data is absent in the amplification plot but visible in the plate view, and the X-axis of the amplification is displaying a range of 0-1 cycles,
4. No Template Control (NTC) is labeled as such (for accurate DRn calculations),
5. The choice of dye component should be made correctly before data analysis. Even if the probe is labeled with FAM and VIC is chosen there will be some result but the wrong one,
6. You must save the run before it starts by giving it a name (not leaving as untitled). Also at the end of the run, first save the data before starting to analyze,
7. The ABI software requires extreme caution. Do not attempt to stop a run after clicking on the Run button. You will have problems and if you need to switch off and on the machine, you have to wait for at least an hour to restart the run.
When analyzing the data, remember that the default setting for baseline fluorescence calculation is cycles 3 - 15 (called baseline cycles). If any CT value is <15, href="http://www.appliedbiosystems.com/support/tutorials/pdf/data_analysis_7700.pdf">Setting Baselines and Thresholds . (Interestingly, this issue is best discussed in the manual for TaqMan Human Endogenous Control Plate.)
If the results do not make sense, check the raw spectra for a possible CDC camera saturation during the run. Saturation of CDC camera may be prevented by using optical caps rather than optical adhesive cover. It is also more likely to happen when SYBR Green I is used, when multiplexing and when a high concentration of probe is used.
For operating instruction of Stratagene Mx3000™ Multiplex Quantitative PCR System, see User Guide and FAQs.
For manuals and other educational material about Idaho Technology Rapid Cycler Instrument, see Support Page.

Interpretation of results
At the end of each reaction, the recorded fluorescence intensity is used for the following calculations by the software of the system used:
Rn+ is the Rn value of a reaction containing all components (the sample of interest); Rn- is the Rn value detected in NTC (baseline value). DRn is the difference between Rn+ and Rn-. It is an indicator of the magnitude of the signal generated by the PCR. It is the DRn plotted against cycle numbers that produces the amplification curves and gives the CT value.

There are different approaches to quantitate the amount of template 50 (Livak, 2001):
1. Absolute standard curve method: Absolute quantification determines the input copy number of the transcript of interest, usually by relating the PCR signal to a standard curve. In this method, a standard curve is first constructed from an RNA of known concentration. This curve is then used as a reference standard for extrapolating quantitative information for mRNA targets of unknown concentrations. cDNA plasmids are the preferred standards for absolute quantitation. This method has been used to estimate cytokine concentrations 51 (Giulietti, 2001), CMV 52-55 (Kearns, 2001a; Kearns, 2001b; Kearns, 2002; Mengelle, 2003), HIV 56 (Gibellini, 2004) and other viral loads 57 (Niesters, 2001), 16 (Saha, 2001). See Bustin, 2000 for a review 39; and Absolute Quantification Page by Pfaffl.
2. Relative standard method (relative fold change): In this method, one of the experimental samples is the calibrator, or 1x sample. Each of the normalized target values is divided by the calibrator normalized target value to generate the relative expression levels. Target quantity is determined from the standard curve and divided by the target quantity of the calibrator. The calibrator is the 1x sample, and all other quantities are expressed as an n -fold difference relative to the calibrator. The calibrator is usually the expression level at baseline and the experimental samples are those collected after treatment or some intervention. The calibrator should be available at large enough quantities to be included in each run. See Relative Quantification Page by Pfaffl.
3. Comparative threshold (CT) method: This method uses no known amount of standard but compares the relative amount of the target sequence to any of the reference values chosen and the result is given as relative to the reference value (such as the expression level of resting lymphocytes or a standard cell line or in comparison to the baseline value). For the CT calculation to be valid, the efficiency of the target amplification and the efficiency of the reference amplification must be approximately equal. A sensitive method for assessing if two amplicons have the same efficiency is to look at how CT varies with template dilution. Before using the DDCT method for quantitation, a validation experiment is performed to demonstrate that efficiencies of target and reference are approximately equal. Serial dilutions of the target and normalizer are prepared and real-time PCR is run in separate tubes. The CT values for each dilution of the target and the normalizer are obtained and their difference for each dilution is calculated (DCT). Then, a plot of log input (like from 0.01 ng to 1 ng) amount versus DCT is prepared. If the efficiencies of the two amplicons are approximately equal, the plot of log input amount versus DCT has a slope of approximately zero (the absolute value of the slope of log input amount vs CT should be < href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12352889">Sabek, 2002). See Livak & Schmittgen, 2001 for a review 50; and ABI-7700 User Bulletin #2 for the details of quantitation methods.

The comparative CT method (DDCT) for relative quantitation of gene expression
This method enables relative quantitation of template and increases sample throughput by eliminating the need for standard curves when looking at expression levels relative to an active reference control (normalizer). For this method to be successful, the dynamic range of both the target and reference should be similar. A sensitive method to control this is to look at how DCT (the difference between the two CT values of two PCRs for the same initial template amount) varies with template dilution. If the efficiencies of the two amplicons are approximately equal, the plot of log input amount versus DCT will have a nearly horizontal line (a slope of <0.10). This means that both PCRs perform equally efficiently across the range of initial template amounts. If the plot shows unequal efficiency, the standard curve method should be used for quantitation of gene expression. The dynamic range should be determined for both (1) minimum and maximum concentrations of the targets for which the results are accurate and (2) minimum and maximum ratios of two gene quantities for which the results are accurate. In conventional competitive RT-PCR, the dynamic range is limited to a target-to-competitor ratio of about 10:1 to 1:10 (the best accuracy is obtained for 1:1 ratio). The real-time PCR is able to achieve a much wider dynamic range.

Running the target and endogenous control amplifications in separate tubes and using the standard curve method requires the least amount of optimization and validation. The advantage of using the comparative CT method is that the need for a standard curve is eliminated (more wells are available for samples). It also eliminates the adverse effect of any dilution errors made in creating the standard curve samples.

As long as the target and normalizer have similar dynamic ranges, the comparative CT method (DDCT method) is the most practical method. It is expected that the normalizer will have a higher expression level than the target (thus, a smaller CT value). The calculations for the quantitation start with getting the difference (DCT) between the CT values of the target and the normalizer:

DCT = CT (target) - CT (normalizer/calibrator/reference)

This value is calculated for each sample to be quantitated (unless, the target is expressed at a higher level than the normalizer, this should be a positive value. It is no harm if it is negative). One of these samples should be chosen as the reference (baseline) for each comparison to be made. The comparative DDCT calculation involves finding the difference between each sample's DCT and the baseline's DCT. If the baseline value is representing the minimum level of expression, the DDCT values are expected to be negative (because the DCT for the baseline sample will be the largest as it will have the greatest CT value). If the expression is increased in some samples and decreased in others, the DDCT values will be a mixture of negative and positive ones. The last step in quantitation is to transform these values to absolute values. The formula for this is:

comparative expression level = 2 - DDCt

For expressions increased compared to the baseline level this will be something like 23 = 8 times increase, and for decreased expression it will be something like 2-3 = 1/8 of the reference level. Microsoft Excel can be used to do these calculations by simply entering the CT values (there is an online ABI tutorial on the use of spread sheet programs to produce amplification plots; the TaqMan Human Endogenous Control Plate protocol also contains detailed instructions on using MS Excel for real-time PCR data analysis). A more accurate method of relative quantification using the relative expression ratio is presented by Pfaffl 58 (Pfaffl, 2001).

The quantification methods are outlined in the ABI User Bulletins. The Bulletins #2 and #5 are most useful for the general understanding of real-time PCR and quantification.

Points to remember and trouble shooting
1. TaqMan Universal PCR master mix should be stored at 2 to 8 0C (not at -20 0C),
2. The GAPDH probe supplied with the TaqMan Gold RT-PCR kit is labeled with a JOE reporter dye, the same probe provided within the Pre-Developed TaqMan Assay Reagents (PDAR) kit is labeled with VIC. Primers for these human GAPDH assays are designed not to amplify genomic DNA,
3. The carryover prevention enzyme, AmpErase UNG, cannot be used with one-step RT-PCR which requires incubation at 48 0C but may be used with the EZ RT-PCR kit,
4. It is ideal to run duplicates to control pipeting errors but this inevitably increases the cost,
5. If multiplexing, the spectral compensation option (in Advanced Options) should be checked before the run,
6. Normalization for the fluorescent fluctuation by using a passive reference (ROX) in the reaction and for the amount of cDNA/PCR efficiency by using an endogenous control (such as GAPDH, active reference) are different processes.
7. Real-time PCR can be used not only for qPCR but also for end-point PCR. The latter includes presence/absence assays (as in pathogen detection) and allelic discrimination assays (SNP genotyping) (see ABI User Guide),
8. Shifting Rn values during the early cycles (cycle 0-5) of PCR means initial disequilibrium of the reaction components and does not affect the final results as long as the lower value of baseline range is reset,
9. If an abnormal amplification plot has been noted (CT value <15 cycles with amplification signal detected in early cycles), the upper value of the baseline range should be lowered and the samples should be diluted to increase the CT value (a high CT value may also be due to contamination),
10. A small DRn value (or greater than expected CT value) indicates either poor PCR efficiency or low copy number of the target. This may also occur in the case of contamination of NTC,
11. A standard deviation >0.16 for CT value indicates inaccurate pipetting,
12. SYBR Green entry in the Pure Dye Setup should be abbreviated as "SYBR" in capitals. Any other abbreviation or lower case letters will cause problems,
13. The ABI 7700 should not be deactivated for extended periods of time. If it has ever been shutdown, it should be allowed to warm up for at least one hour before a run. Leaving the instrument on all times is recommended and is beneficial for the laser. If the machine has been switched on just before a run, an error box stating a firmware version conflict may appear. If this happens, choose the "Auto Download" option,
14. The ABI 7700 (or its successor 7900) is only one of the many real-time PCR systems in a very competitive market (see also reviews by Bustin SA, 2000, Bustin SA, 2002; 39,59 Supplier Guide by Bonetta, 2005; Biocompare; Gene-Quantification Site).

See also qPCR tips and troubleshooting at qPCR Troubleshooting Guide by ABgene; Protocol Online qPCR troubleshooting; Ten Most Common Real-Time PCR Pitfalls; Stratagene FastTrack Basic Assay Troubleshooting.

Advantages of using Real-Time PCR
* Traditional PCR is measured at end-point (plateau), while real-time PCR collects data in the exponential growth phase
* An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated
* The cleaved probe provides a permanent record amplification of an amplicon
* Increased dynamic range of detection
* Requirement of 1000-fold less RNA than conventional assays
* No-post PCR processing due to closed system (no electrophoretical separation of amplified DNA)
* Detection is capable down to a 2-fold change
* Small amplicon size results in increased amplification efficiency

Real-Time PCR Applications
Real-Time PCR can be applied to traditional PCR applications as well as new applications that would have been less effective with traditional PCR. With the ability to collect data in the exponential growth phase, the power of PCR has been expanded into applications such as:
* Copy number variation (CNV): (ABI TaqMan® Gene Copy Number Assays; Protocol for 7900HT)
* Quantitation of gene expression (Giulietti, 2001) including NK cell KIR gene expression 60 (Leung, 2005)
* Array verification 61 (Rajeevan, 2001). See also Verification of Array Results Page by Pfaffl.
* Biosafety and genetic stability testing 62 (Lovatt, 2002)
* Drug therapy efficacy / drug monitoring 63 64 65 66 (Leruez-Ville, 2004; Brennan, 2003; Burger, 2003; Kogure, 2004)
* Real-Time Immuno-PCR (IPCR) 67-69 (Adler, 2003; Barletta, 2004; Lind & Kubista, 2005)
* Chromatin Immunoprecipitation (ChIP) 70-75 (Braveman, 2004; Sandoval, 2004; Wang, 2004; Iype, 2005; Potratz, 2005; Puppo, 2005)
* Viral quantitation 55,57 (Niesters, 2001; Mengelle, 2003)
* Pathogen detection 76-81 (Belgrader, 1999; Uhl, 2002; Mackay, 2004; Perandin, 2004; Watzinger, 2004; Reynisson, 2006) including CMV detection 52-55 (Kearns, 2001a; Kearns, 2001b; Kearns, 2002; Mengelle, 2003), rapid diagnosis of meningococcal infection 82 (Bryant, 2004), penicillin susceptibility of Streptococcus pneumoniae 83 (Kearns, 2002), Mycobacterium tuberculosis and its resistant strains 84-87 (Kraus, 2001; Torres, 2003; Cleary, 2003; Hazbon, 2004), and waterborne microbial pathogens in the environment 88,89 (Foulds, 2002; Guy, 2003)
* DNA damage (microsatellite instability) measurement 90 (Dietmaier, 2001)
* Radiation exposure assessment 28,91-93 (Blakely, 2001; Blakely, 2002; Grace, 2002; Grace, 2003)
* In vivo imaging of cellular processes 94,95 (Tung, 2000; Bremer, 2002)
* Mitochondrial DNA studies 96-98 (He, 2002; Liu, 2003; Alonso, 2004)
* Methylation detection 99-102 (Trinh, 2001; Cottrell, 2004; Lehmann & Kreipe, 2004; Thomassin, 2004)
* Detection of inactivation at X-chromosome 103,104 (Hartshorn, 2002; van Dijk, 2002)
* Determination of identity at highly polymorphic HLA loci 105 (Zhou, 2004)
* Monitoring post transplant solid organ graft outcome 43,44 (Sabek, 2002; Gibbs, 2003)
* Monitoring chimerism after hematopoietic stem cell transplantation 106-109 (Elmaagacli, 2002; Alizadeh, 2002; Thiede, 2004; Harries, 2004)
* Monitoring minimal residual disease after hematopoietic stem cell transplantation 9,106,110-112 (Elmaagacli, 2002; Cilloni, 2002; Sarris, 2002; Gabert, 2003; Van der Velden, 2003)
* Determination of gene dosage and zygosity 113-115 (Bubner, 2004; Barrois, 2004; Chen, 2006; Szilagyi, 2006)
* Genotyping by fluorescence melting-curve analysis (FMCA) or high-resolution melting analysis (HRMA) 26,116-123 (von Ahsen 2000; Donohoe, 2000; Lyon, 2001; Waterfall & Cobb, 2002; Bennett, 2003; Wittwer, 2003; Zhou, 2005; Palais, 2005; Chou, 2005) or specific probes/beacons 11,17,124-127 (Tapp, 2000; Mhlanga, 2001; Solinas, 2001; Song, 2002; Gupta, 2004; reviewed in Lareu, 2004)
- Trisomies 128 (Zimmermann, 2002) and single-gene copy numbers 129-132 (Bieche, 1998; Mocellin, 2003, Barrois, 2004; Linzmeier, 2005).
- Microdeletion genotypes 133-136 (Laurendeau, 1999; Kariyazono, 2001; Covault, 2003; Coupry, 2004)
- Haplotyping 137,138 (Von Ahsen, 2004; Pont-Kingdon & Lyon, 2005)
- Quantitative microsatellite analysis 139 (Ginzinger, 2000)
- DNA pooling and quantitative allelic discrimination 140-142 (Barcellos, 2001; Abbas, 2004; Quesada, 2004)
- Prenatal diagnosis / sex determination using single cell isolated from maternal blood 143-145 (Hahn, 2000; Bischoff, 2002; Bischoff, 2003) or fetal DNA in maternal circulation 144,146 (Bischoff, 2002; Hwa, 2004)
- Prenatal diagnosis of hemoglobinopathies 29,147,148 (Kanavakis, 1997; Vrettou, 2003; Vrettou, 2004)
- Intraoperative cancer diagnostics 149 (Raja, 2002)
* Linear-after-the-exponential (LATE)-PCR: a new method for real-time quantitative analysis of target numbers in small samples, which is adaptable to high throughput applications in clinical diagnostics, biodefense, forensics, and DNA sequencing 150 (Sanchez, 2004).

Full References Cited Automated PubMed Search for Real-Time PCR
New Publications

Quantitative PCR Gene Expression Profiling by MultiD - Tutorials
TATAA Biocenter Open Courses in Quantitative PCR
Workshops and Courses by Stephen Bustin

Internet links
Idaho Technology RapidCycler User Support Literature
ROCHE LightCycler Online LightCycler University Products for LightCycler
Bio-Rad iCycler Stratagene Multiplex qPCR System & qPCR Guide Corbett Research Rotor-Gene
Cepheid Smart Cycler BioGene InSyte Techne Quantica MJ Research Real-Time Systems Eppendorf Mastercycler
Applied Biosystems Sequence Detection Systems
ABI User Bulletins ABI-PRISM 7700 Application Notes 7900HT 7000 Compendium
SNP500Cancer Validated Allelic Discrimination Assay List (including TaqMan Protocols)
Available Real-Time PCR Platforms BioCompare
1st International qPCR Symposium & Application Workshop

Scorpion Technology Molecular Beacons Light-Up Probes (1) (2)
D-LUX Designer LNA Probes LNA Primers (Exiqon OligoDesign) Exiqon ProbeLibrary
TaqMan Gene Expression Assays
Primer-Probe Design Tool (OligoSys) by TATAA BioCenter
Primer-Probe and Beacon Design Program & Demo by Premier
RT-PCR PrimerBank RT-PCR Primer DataBase RT-PCR Primer Sets
AlleleID Pathogen Detection Primer & Probe Design Tool by Premier Biosoft International
Quantitative PCR Primer Database - QPPD (NCI)
PrimerDesign
Frequently Asked Questions (Real-Time PCR Primers)

Transcript of a Webcast on Real-Time PCR Applications (Bio.Com)
Biocompare Tutorials:
Tools and Technologies for Real-Time PCR & Fast PCR (text)
STRATAGENE Online qPCR Training
Roche LightCycler Literature and Technical Notes
QiaGen Handbooks on SYBR Green Detection Systems and RT-PCR
Ambion TechNotes on Real-Time PCR
Full qPCR Protocol (Nolan, Hands & Bustin, Nature Protocols, 2006) (PDF)
A-Z Quantitative PCR by Stephen Bustin
Gene Quantification Page by Michael W Pfaffl & Directory Page
Real-Time PCR Tutorial (South Carolina University)
Real-Time PCR: Short Course (University of Texas)
Real-Time PCR Handbook (University of Illinois at Chicago)
Real-Time PCR Seminar (NIEHS) & Review by Nigel Walker
Real-Time PCR in Infectious Diseases (PPT) & (PDF) by Theo Sloots
Real Time PCR & Quantitation Lecture by Ian MacKay
Q-PCR Training @ TATAA BioCenter
Molecular Probes Handbook: Nucleic Acid Detection and Genomics Technology
PCR and Real Time PCR Links Real-Time PCR Literature
Links at PCRlinks.com Links at Protocol Online
Real Time PCR Special Issue (Dec 2001, Vol.25, Issue 4) of METHODS Journal
Review of real-time PCR in mRNA Quantitation (Wong & Medrano, 2005)
Five Questions on qPCR & How It Works (The Scientist)

Statistics and Gene Expression Analysis BioInformatics in Real-Time PCR
Q-GENE for data processing
qBASE geNORM
BestKeeper© for determination of stable housekeeping genes (Download)
REST© for Relative Expression Software Tool (Download / Corbett)
Peirson, 2003 (DART-PCR) (Download)

Address for bookmark: http://www.dorak.info/genetics/realtime.html

M.Tevfik Dorak, MD, PhD
Last updated on 24 November 2007
Genetics Evolution HLA MHC Biostatistics Glossary Homepage

The Fast And The Furry

2007-12-03T11:45:00.000-05:00

كارتون جذاب تام و جري كه ديدنش بزرگ و كوچيك نداره ،The Fast And The Furry
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http://rapidshare.com/files/۲۹۶۰۶۹۶۷/Tom_Jerry-The.Fast.and.The.Furry.part۴.rar

http://rapidshare.com/files/۲۹۶۱۲۱۵۵/Tom_Jerry-The.Fast.and.The.Furry.part۵.rar

http://rapidshare.com/files/29612297/Tom_Jerry-The.Fast.and.The.Furry.part6.rar

كارتون

2007-11-18T22:44:00.000-05:00

كارتون های دوبله شده توسط گروه گلوری یه مزه دیگه ای دارن، حتماً شما هم دوست دارید آرشیوی از این كارتون ها رو داشته باشید

شیرشاه یك و نیم- Lion King 1½

Total of 619MB, video coded with XviD codec, 2xMP3 coded audio streams: 1-Farsi 2-English

Rapidshare: Part1---------MegaUploads: Part1
Rapidshare: Part2---------MegaUploads: Part2 -
Rapidshare: Part3-------- MegaUploads: Part3 -
apidshare: Part4----------MegaUploads: Part4
Rapidshare: Part5 -------- MegaUploads: Part5
Rapidshare: Part6 -------- MegaUploads: Part6
Rapidshare: Part7 -------- MegaUploads: Part7
Rapidshare: Part8 -------- MegaUploads: Part8
Rapidshare: Part9 -------- MegaUploads: Part9

رئیس مزرعه - Barnyard

Total of 612MB, video coded with XviD codec, 2xMP3 coded audio streams: 1-Farsi 2-English

Rapidshare: Part1 -------- MegaUploads: Part1
Rapidshare: Part2 -------- MegaUploads: Part2
Rapidshare: Part3 -------- MegaUploads: Part3
Rapidshare: Part4 -------- MegaUploads: Part4
Rapidshare: Part5 -------- MegaUploads: Part5
Rapidshare: Part6 -------- MegaUploads: Part6
Rapidshare: Part7 -------- MegaUploads: Part7
Rapidshare: Part8 -------- MegaUploads: Part8
Rapidshare: Part9 -------- MegaUploads: Part9

Chicken Little - جوجه كوچولو

Total of 633MB, video coded with XviD codec, 2xMP3 coded audio streams: 1-Farsi 2-English
Part01
Part02
Part03 Reported BAD or DEAD by 1 User(s) --->--Biguploads/Part3 Mirror at Biguploads
Warning: if you've downloaded Part3 from Bigupload, rename (replace under lines with space) to achive the correct file name.
Part04
Part05
Part06
Part07
Part08
Part09
Part10

Dragon Blade - شمشیر اژدها

Total of 523MB, video coded with XviD codec, 1xMP3 coded audio stream:
Part01
Part02
Part03
Part04
Part05
Part06
Part07
Part08

Anastasia - آناستازیا
Total of 645MB, video coded with XviD codec, 2xMP3 coded audio streams: 1-Farsi 2-
Part01
Part02
Part03
Part04
Part05
Part06
Part07
Part08
Part09
Part10

Garfield The Movie - گارفیلد

Total of 680MB, video coded with DivX codec, 2xMP3 coded audio streams: 1-Farsi 2-English
Part01
Part02
Part03
Part04
Part05
Part06
Part07
Part08
Part09
Part10

Apple Seed - دانه سیب

Total of 620MB, video coded with DivX codec, 1xMP3 coded audio stream: Farsi
Part01
Part02
Part03
Part04
Part05
Part06
Part07
Part08
Part09

Casper's Haunted Christmas - كسپر در كریسمس روح زده

Total of 652MB, video coded with DivX codec, 2xMP3 coded audio streams: 1-Farsi 2-English

Part01
Part02
Part03
Part04
Part05
Part06
Part07
Part08
Part09
Part10

USB Ubuntu 7.10 Gutsy Gibbon install

2007-10-25T13:54:00.000-04:00

USB Ubuntu 7.10 install from Linux: This tutorial enables you to install, boot and run Ubuntu 7.10 (Gutsy Gibbon) from USB. In addition to installing Ubuntu to a USB device and then booting Ubuntu from USB, this tutorial will enable you to automatically save your changes and settings back to the stick and further restore them on each boot using a second "casper-rw" persistent partition. The tutorial was written for those already familiar with working from Ubuntu or another Linux desktop environment. If you do not have access to or prefer not to use a Windows computer, this Ubuntu Linux on a stick tutorial is for you.
Ubuntu 7.10 takes slightly longer to boot than previous releases. However, once it's up and running, it performs much better than running from the Live CD.
USB Ubuntu 7.10 Essentials:
Ubuntu7.10 ISO
CD Burner
1GB USB flash drive (2GB+ recommended)
U710fix.tar
Ubuntu 7.10 USB installation tutorial:
Hint: You can drastically speed up the install by Copying (Ctrl+c) and Pasting (Ctrl+v) commands into the terminal instead of manually typing them out. With the exception of replacing X with your drive letter.
Grab the Ubuntu 7.10 ISO and burn it to a CD
Insert the CD and your USB flash drive
Reboot your computer into Ubuntu from the Live CD
Open a terminal window and type sudo su
Type fdisk -l to list available drives/partitions. Note which device is your flash drive (example: /dev/sda) Throughout this tutorial, replace x with your flash drive letter. For example, if your flash drive is sdb, replace x with b.
Type umount /dev/sdx1
Type fdisk /dev/sdx
type p to show the existing partition and d to delete it
type p again to show any remaining partitions (if partitions exist, repeat the previous step)
type n to make a new partition
type p for primary partition
type 1 to make this the first partition
hit enter to use the default 1st cylinder
type +750M to set the partition size
type a to make this partition active
type 1 to select partition 1
type t to change the partition filesystem
type 6 to select the fat16 file system
type n to make another new partition
type p for primary partition
type 2 to make this the second partition
hit enter to use the default cylinder
hit enter again to use the default last cylinder
type w to write the new partition table
Type umount /dev/sdx1 to ensure the 1st partition is unmounted
Type mkfs.vfat -F 16 -n ubuntu710 /dev/sdx1 to format the first partition
Type umount /dev/sdx2 just to ensure the 2nd partition is unmounted
Type mkfs.ext2 -b 4096 -L casper-rw /dev/sdx2 to format the second partition
Remove and Re-insert your flash drive
Back at the terminal, type apt-get update
Type apt-get install syslinux mtools
Type syslinux -sf /dev/sdx1
Type cd /cdrom
Type cp -rf casper disctree dists install pics pool preseed .disk isolinux/* md5sum.txt README.diskdefines ubuntu.ico casper/vmlinuz casper/initrd.gz /media/ubuntu710/
Ignore any "cannot create symbolic link" errors
Type cd /home/ubuntu
Type wget pendrivelinux.com/downloads/U710fix.zip
Type unzip -o -d /media/ubuntu710/ U710fix.zip
Restart your computer, set your BIOS or Boot menu to boot from the USB device and reboot again.
You should now have a USB Ubuntu 7.10 Gutsy Gibbon flash drive that should automatically save your changes, restoring them on boot.
Note: If your having trouble getting Ubuntu to boot, your memory stick may have a corrupted MBR. To repair the MBR of your USB device, at the terminal type sudo apt-get install lilo then type lilo -M /dev/sdx (replacing x with the letter of your flash device)

آموزش زبان

2007-10-25T13:36:00.000-04:00

سارا هر روز یک ویدئو آموزشی در حد چند دقیقه آماده و در یوتیوب آپلود می کند. . درون هر پست می توانید لینک ویدئو مربوطه را ببینید و تمامی گفته هایی که درون ویدئو گفته می شود را مطالعه کنید. پس اگر متوجه کلمه یا کلماتی نشوید می توانید به راحتی متن موجود درون وبلاگ را بخوانید
هر روز با یک مطلب جدید (نه کمتر و نه بیشتر!) در اختیار بازدید کنندگان خواهد بود

با امکانات این سایت به راحتی ویدئوهای سارا را دانلود و با خیال راحت به تماشای آنها بنشینید

Windows Vista Live CD

2007-10-25T13:16:00.000-04:00

Windows Vista Live CD !Just Burn / Boot and Enjoy

Useful for system crash recovery

Just insert disk in your CD Rom And boot Form CD

There in No need To instal , run driect from CD

File Size: 110MB

Checked & Tested: Yes

Format: .ISO File

Parts: 4

Download: Rapidshare
http://rapidshare.com/files/32882575/ViSTA.Live-SHiMLA.part1.rar

http://rapidshare.com/files/32882580/ViSTA.Live-SHiMLA.part2.rar

http://rapidshare.com/files/32882587/ViSTA.Live-SHiMLA.part3.rar

http://rapidshare.com/files/32882534/ViSTA.Live-SHiMLA.part4.rar

Mirror: Direct Links: Mihd.net

Code:

http://mihd.net/apw49l

http://mihd.net/0nqtev

http://mihd.net/681z92

http://mihd.net/e9buho

Can you read this text?

2007-10-19T14:46:00.000-04:00

If yuo cna raed tihs, yuo hvae a sgtrane mnid too.Olny 55% fo plepoe cna.

i cdnuolt blveiee taht I cluod aulaclty uesdnatnrd waht I was rdanieg. The phaonmneal pweor of the hmuan mnid, aoccdrnig to a rscheearch at Cmabrigde Uinervtisy, it dseno’t mtaetr in waht oerdr the ltteres in a wrod are, the olny iproamtnt tihng is taht the frsit and lsat ltteer be in the rghit pclae. The rset can be a taotl mses and you can sitll raed it whotuit a pboerlm. Tihs is bcuseae the huamn mnid deos not raed ervey lteter by istlef, but the wrod as a wlohe. Azanmig huh? yaeh and we awlyas tghuhot slpeling was ipmorantt!

روایتی از “سرود ملی” ایران

2007-10-19T14:27:00.000-04:00

(داستان زير مربوط به دانشجويان ايراني است که دوران سلطنت احمدشاه قاجار براي تحصيل به آلمان رفته بودند و از قول آقاي دکتر جلال گنجي فرزند مرحوم سالار معتمد گنجي نقل شده است.)
ما هشت دانشجوي ايراني بوديم که در آلمان در عهد احمد شاه تحصيل مي‌کرديم. روزي رئيس دانشگاه به ما اعلام نمود که همۀ دانشجويان خارجي بايد از مقابل امپراطور آلمان رژه بروند و سرود ملي کشور خودشان را بخوانند. ما بهانه آوريم که عده مان کم است. گفت: اهميت ندارد. از برخي کشورها فقط يک دانشجو در اينجا تحصيل مي‌کند و همان يک نفر، پرچم کشور خود را حمل خواهد کرد، و سرود ملي خود را خواهد خواند.
چاره ای نداشتيم. همۀ ايراني‌ها دور هم جمع شديم و گفتيم ما که سرود ملي نداريم، و اگر هم داريم، ما به ‌ياد نداريم. پس چه بايد کرد؟ وقت هم نيست که از نيشابور و از پدرمان بپرسيم. به راستي عزا گرفته بوديم که مشکل را چگونه حل کنيم. يکي از دوستان گفت: اينها که فارسي نمي‌دانند. چطور است شعر و آهنگي را سر هم بکنيم و بخوانيم و بگوئيم همين سرود ملي ما است. کسي نيست که سرود ملي ما را بداند و اعتراض کند.
اشعار مختلفي که از سعدي و حافظ مي‌دانستيم، با هم تبادل کرديم. اما اين شعرها آهنگين نبود و نمي‌شد بصورت سرود خواند. بالاخره من (دکتر گنجي) گفتم: بچه‌ها، عمو سبزي ‌فروش را همه بلديد؟ گفتند: آري. گفتم: هم آهنگين است، و هم ساده و کوتاه. بچه‌ها گفتند: آخر عمو سبزي ‌فروش که سرود نمي‌شود. گفتم: بچه‌ها گوش کنيد! و خودم با صداي بلند و خيلي جدي شروع به خواندن کردم:
“عمو سبزي ‌فروش . . . بله. سبزي کم‌فروش . . . بله. سبزي خوب داري؟ . . . بله.”
فرياد شادي از بچه‌ها برخاست و شروع به تمرين نموديم. بيشتر تکيۀ شعر روي کلمۀ”بله” بود که همه با صداي بم و زير مي‌خواندیم.

:همۀ شعر را نمي‌دانستيم. با توافق همديگر، “سرود ملي” به اين‌صورت تدوين شد

عمو سبزي‌فروش! . . . بله

سبزي کم‌فروش! . . . . بله

سبزي خوب داري؟ . .. بله

خيلي خوب داري؟ . . . بله

عمو سبزي‌فروش! . . . بله

سيب کالک داري؟ . . . بله

زال‌زالک داري؟ . . . . بله

سبزيت باريکه؟ . . . . بله

شبهات تاريکه؟ . . . . بله
عمو سبزي‌فروش! . . . بله

اين را چند بار تمرين کرديم. روز رژه، با يونيفورم یک ‌شکل و یکرنگ از مقابل امپراطور آلمان، “عمو سبزي ‌فروش” خوانان رژه رفتيم. پشت سر ما دانشجويان ايرلندي در حرکت بودند. از “بله” گفتن ما به هيجان آمدند و “بله” را با ما همصدا شدند، به‌ طوري که صداي “بله” در استاديوم طنين‌انداز شد و امپراطور هم به ما ابراز تفقد فرمودند و داستان به‌خير گذشت.
منبع:
فصلنامۀ “ره‌ آورد” شمارۀ 35، صفحۀ 286 – 287
اطلاعات بیشتر:
تاريخچۀ “سرود ملي” در ايران (سایت پرند)
نماهنگ (swf) اولین “سرود ملی” ایران (ایران کلیپ)
تاريخچۀ “سرود ملي” در ايران (آهنگ زمانه)

تشخیص سرطان سینه در مراحل اولیه

2007-10-19T14:21:00.000-04:00

همه ما از اینکه خواهر، مادر، اقوام، دوستان و یا آشنایانمان در اثر ابتلا به سرطان سینه قسمتی از بدن و یا حتی جان خود را از دست می دهند بسیار متاثر می شویم؟ آیا با دانستن این حقیقت که با انجام چند کار ساده و بدون هزینه می توان سرطان سینه را در مراحل اولیه تشخیص و بطور موثری در مان کرد، بیش از پیش متاسف نخواهید شد؟ اگر چه تاسف گذشته را عوض نمی کند اما شاید با خواندن مقاله زیر جایی برای تاسف در آینده باقی نماند. سرطان سینه شایع ترین و کشنده ترین نوع سرطان در زنان است. در ایران درصد بالایی از زنان مبتلا شده به سرطان سینه جان خود را از دست میدهند زیرا سرطان سینه آنان در مراحل اولیه تشخیص داده نشده اند. مطالب زیر که از چندین مرجع معتبر اقتباس و ترجمه شده اند، به بیان روشی ساده برای کمک به تشخیص اولیه بسیاری از انواع سرطان های سینه می پردازد که میتواند تن و یا جان زنان را نجات دهد.
معاینه شخصی سینه: Breast Self Exam (BSE)
معاینه شخصی سینه یکی از راههای مهم، برای تشخیص زود هنگام سرطان سینه است. البته تمام انواع سرطان های سینه با معاینه شخصی تشخیص داده نمی شوند اما این اولین قدمی است که شما می توانید و باید برای سلامتی خودتان بردارید. بسیاری از خانم ها ممکن است تمایلی به معاینه سینه خود نداشته باشند زیرا نگرانند چیزی را لمس کند که نمی دانند چیست، اما هر چه بیشترمعاینه شخصی را تکرار کنید بیشتر درباره آن می دانید و بهتر متوجه تغییرات غیر طبیعی آن می شوید.
سعی کنید BSE را یکبار در ماه انجام دهید تا به شکل و وضعیت طبیعی سینه خود پی ببرید. BSE را چند روز بعد از پایان قاعدگی انجام دهید زیرا در آن زمان سینه ها کمترین حجم و تورم را دارند. اگر شما دوران یائسگی را طی می کنید، روزی را انتخاب کنید که به راحتی در خاطرتان بماند، مثلاً اول یا آخر ماه. اگر توده ای (جسم سفتی) را در سینه خود یافتید، وحشتزده نشوید. بیشتر زنان همیشه چند توده و یا مناطق تو دهای در سینه خود دارند. خبر خوب این است که از هر10 توده ای که از سینه برداشته می شود، هشت تای آن بی خطر و غیر سرطانی هستند!
قسمت بالای سینه که به زیر بغل نزدیک است مهمترین غدد را در بر می گیرد. نیمه دیگر پایینی سینه، مانند ماسه ساحل احساس می شود. قسمت زیر نوک سینه را می توان بصورت مجموعه ای شبیه دانه هایی درشت احساس کرد. دیگر قسمت آن مانند یک کاسه گندمبا احساس می شود. نکته مهم این است که شما با شکل این برجستگی های مختلف سینه خود آشنا شوید تا اگر به توده غیر عادی برخورد کردید ( مانند سنگی بر شنهای ساحل) و اگر چیزی در سینه شما تغییر کرده، توجه دکتر خود را به آن جلب کنید.
دانستن اینکه سینه شما معمولاً چطور به نظر می سد و احساس می شود، میتواند شما را از دردسر نمونه برداری توسط دکتر (biopsy برداشت قسمتی از بافت سینه و بررسی آن در زیر میکروسکوپ) مصون دارد.
یادداشت برداری مفید است!
بخصوص در ماههای اول که ممکن است مشخصات سینه را در ماه قبل فراموش کنید، با یادداشت برداری و کشیدن نقشه ای وضعیت سینه خود را ثبت کنید. در جاهای که غدد غیر معمولی را احساس می کنید، در یادداشت خود علامت بگذارید. غیر طبیعی نیست اگر توده ای در روز های خاصی در ماه احساس شوند و بعد ناپدید گردند زیرا بدن شما با قاعدگی تغییر می کند. فقط تغییراتی که بیش از دوره قاعدگی طول می کشند و یا غددی که بزرگتر می شوند و یا به نوعی خصوصیت مشخصی دارند، را باید به پزشکتان گزارش کنید.
روش معاینه شخصی سینه
برای اینکه معاینه شخصی سینه بصورت مفید و کارایی انجام شود باید آنرا با دقت و توجه انجام داد که در زیر به مراحل مختلف آن اشاره میکنیم.
مرحله اول: در این مرحله، بدن میتواند در حالت دراز کش روی زمین و یا ایستاده در زیر دوش باشد. در یکی از این حالت ها باید به ترتیب زیر سینه خود را معاینه کنید:
1. دست راست خود را در پشت سر خود قرار دهید و از قسمت نرم سه انگشت میانه دست چپ خود، برای معاینه سینه راست خود استفاده کنید. ( اگر در زیر دوش هستید از آب صابون نیز می توانید برای بهتر لمس شدن آن استفاده کنید.)
2. انگشتانتان را بر روی سینه خود، با فشار های کم، معمولی و محکم، همانطور که در شکل آمده، بصورت حرکات دایره ای و بدنبال آن حرکات بالا و پایین و سپس با حرکات کششی باز و بسته بر سینه حرکت دهید.
3. این کار را برای سینه سمت چپ خود نیز تکرار کنید.
مرحله دوم: این مرحله باید سینه خود را در مقابل آینه معاینه کنید و بدنبال تغییری در شکل، اندازه و ظاهر سینه خود باشید. به عنوان مثال، فرو رفتگی یا جمع شدگی، کشیدگی، قرمز رنگی و یا مشاهده ترشح چرکی از سر سینه و هر تغییری که به طورعادی آنرا مشاهده نمی کنید. سینه خود را در حالت های زیر مشاهده کنید:
1. دو دست را در طرفین بدن بگیرید.
2. دو دست را در بالای سر بگیرید.
3. دو دست را بر روی باسن فشار دهید تا سینه محکم شود.
4. د رحالیکه دستانتان بر روی باسنتان قرار دارد، به جلو خم شوید.
معاینه سالانه سینه توسط پزشک
معاینه سینه باید بخشی از معاینه سالانه شما توسط دکتران باشد. برخی از زنان از اینکه چرا با وجود اینکه خودشان به معاینه سینه خود می پردازند ولی بازنیاز است که دکتر نیز این کار را بکند، متعجبند. با وجود اینکه بیشتر غدد، با کمک معاینه شخصی خود زنان پیدا می شوند، اما یک معاینه توسط دکتر می تواند غده هایی را که متوجه آنها نشده اید، کشف کند. گاهی تشخیص غیر طبیعی بودن سینه آنقدر مشکل است که تنها یک متخصص قادر به دریافتن آن می باشد. شما ممکن است متوجه تغییراتی نظیر ضخامت و عدم تطابق سینه ها با هم را نشوید، اما کسی که سینه های متعددی را معاینه می کند، متوجه آنها خواهد شد.
مطالعات نشان می دهد ترکیبی از معاینه ماهانه شخصی و معاینه سالانه پزشک، امکان تشخیص سرطان سینه در مراحل اولیه (قابل درمان) را افزایش می دهد.
همانطور که مطلع هستید، متاسفانه فرهنگ آموزش و انجام معاینه شخصی در ایران وجود ندارد در حالیکه این روشی بسیار ساده و در عین حال حیاتی است. با باور این حقیقت که معاینه شخصی نقشی اساسی در تشخیص زود هنگام سرطان داشته و میتواند جانتان را از مرگ نجات دهد، آنرا کاملاً جدی گرفته و از آن غفلت نکنید.
منابع :
Breast Cancer Organization
Breast Self Exam (BSE) Guide
منابع دیگر:
آفتاب: سرطان سینه شایعترین سرطان زنان
ورزش از سرطان سینه جلوگیری می کند
Breast Self Exam (BSE) Brochure
Cancer Health for Young Adults

!چگونه در تنهایی از حمله قلبی جان نسپارید

2007-10-19T14:16:00.001-04:00

تجسم كنيد، ساعت 6 بعد از ظهر است و شما به تنهائي در حال رانندگي به طرف منزل هستيد. بعد از يك روز سخت، شما واقعاً خسته و بي حوصله ايد و اضطراب دارید. ناگهان دردي در قفسه سينه تان شروع مي شود و بازوی چپ و فك پایينتان را نیز فراميگيرد. فقط چند كيلومتر از نزديك ترين بيمارستان دور هستيد ولی مطمئن نیستید که بتوانید خود را در این فاصله سالم به بيمارستان برسانيد. شايد شما ازمتخصصين باشيد و روش هاي پيشگيري از حمله قلبی را بخوبي بدانيد ولي هرگز روي خود انجام نداده باشيد

بيشتر افراد زماني که دچار حملات قلبي مي شوند، تنها و بدون کمک هستنند. فردي كه دچار حمله شده احساس از حال رفتگي مي كند و تا از دست دادن هوشياريش تنها 10 ثانيه فاصله دارد.
نترسید. ماشین را سریعاً کنار بزنید. شروع به سرفه كردن كنيد. بصورت پي در پي و محكم. قبل از هر سرفه يك نفس عميق بكشيد. سرفه بايد عميق و طولاني باشد مثل زماني كه مي خواهيد خلط را ازته سينه خود خارج كنيد. با اورژانس محلی تماس بگیرید و مکان خود را بگویید
نفس عميق و سرفه بايد هر دو ثانیه یکبار و بدون توقف پي در پي تكرار شود تا هنگاميكه كمك برسد و يا قلب شما مجدداً بطور طبیعی بزند. نفس عميق باعث ورود اكسيژن به داخل ريه هاي شما مي شود و سرفه باعث مي گردد به قلب شما فشار وارد شده و سبب چرخش خون شود. اين فشار باعث برگشت آهنگ طبیعی ضربان قلب نيز مي گردد.
فكر نكنيد چون سن شما زير 25 و يا 30 است، شما دچار حملات قلبي نخواهید شد. امروزه به علت تغيير در روش زندگي ما، حملات قلبي بين تمامي سنين مشاهده مي گردد. شما با آگاهی دادن به دوستانتان ممکن است بتوانید جانشان را نجات دهید
:منبع
Attack Heart Attack even if you are alone!
:اطلاعات بیشتر
How to survive a Heart Attack if you are alone!
How a Heart Attack Happens?

یار دبستانی

2007-10-19T14:07:00.000-04:00

ترانه سرا: منصور تهرانی
درسال 1359، زنده یاد فریدون فروغی ترانهٔ بار دبستانی را برای فیلم از فریاد تا ترور به کارگردانی منصور تهرانی اجرا می‌کند که در تیتراژ فیلم استفاده می‌شود. ولی به تهرانی خبر می‌دهند که باید صدای فروغی را از تیتراژ فیلمش حذف کند. در نتیجه جمشید جم خواندن این ترانه را به عهده می‌گیرد. این ترانه در سالهای اخیربسیار معروف شده بطوریکه حتی کاندیدهای انتخاباتی نیز از آن برای جذب جوانان استفاده کرده اند.
یار دبستانی من، با من و همراه منی …………… چوب الف بر سر ما، بغض من و آه منی
حک شده اسم من و تو، رو تن این تخته سیاه … ترکه ی بیداد و ستم، مونده هنوز رو تن ما
دشت بی فرهنگی ما، هرزه تموم علفاش … خوب اگه خوب؛ بد اگه بد، مرده دلای آدماش
دست من و تو باید این پرده ها رو پاره کنه … کی میتونه جز من و تو درد مارو چاره کنه؟

منابع بیشتر:
ترانه “یار دبستانی” با صدای فریدون فروغی (mp3)
ترانه “یار دبستانی” با صدای جمشید جم
مصاحبه با جمشید جم درباره فریدون فروغی
نماهنگ “یار دبستانی” با صدای جمشید جم (swf)
آکوردهای ترانه “یار دبستانی” (pdf)