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.
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