۱۳۸۶ آذر ۲۹, پنجشنبه

اماراتی ها و دیپلم مامایی از گوگل



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

ECG LEARNING CENTER IN CYBERSPACE

ATLAS OF DISEASES OF KIDNEY

ATLAS OF DISEASES OF KIDNEY

Clinical Examination Videos for USMLE CS

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

Obstetric Examination

CPR Video

CPR Video

Physical Examination (Video Clips)

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
__________________

۱۳۸۶ آذر ۲۸, چهارشنبه

Online surgery videos

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...


-----------------------------------------
Lifespan - Online surgery videos


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

MedlinePlus:Videos of Surgical Procedures


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

Cardiothoracic Surgery: University of southern California keck school of medicine


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

The Heart Surgery Forum



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

Spine Universe: spinal surgery


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

Understandspinesurgery.com: spinal surgery procedures


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

Laparoscopy.com


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

Hand Surgery Videos


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

Medicdirect: downloadable surgical operations videos


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

The Royal College Of Surgeons Of Edinburgh:


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

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


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

۱۳۸۶ آذر ۲۷, سه‌شنبه

Making of a PCR Internal Standard

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

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

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

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
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