Chapter 10 - RT-PCR: a science and an art form

Abstract

RT-PCR is a technology by which RNA molecules support the synthesis of complementary DNA (cDNA) sequences by the action of reverse transcriptase (RT), followed by the amplification of the newly synthesized cDNA by standard PCR procedures. This approach to studying gene expression is so named because of the role of RT in the synthesis of first-strand cDNA. RT-PCR is a two-step process. Normalization of samples is very important in RT-PCR, and the efficiency of first-strand cDNA synthesis is one of the most important determinants of the success or failure of this method. For this reason, it is strategically better to make a large cDNA pool from which aliquots may be drawn for individual applications rather than repeating the same cDNA synthesis reaction over and over. The key to RT-PCR resides in the design of well-characterized primers which promote a balance between template specificity, thermodynamic stability when base-paired to the template, and the capacity of one primer to function with the other(s) to support RT-PCR. Given the extreme sensitivity of PCR, it is now commonplace to detect and quantify transcripts present in extremely low abundance. Numerous permutations of RT-PCR are in widespread use, including mapping the 5′ and 3′ ends of transcripts. Hot-start PCR, touchdown PCR, and cloning of PCR products are commonly part of the workflow. RT-PCR is also useful for detecting viral RNA genomes using a one-tube RT-PCR format. There are many non-PCR amplification tools that can be used to verify PCR data if necessary.