Chapter 8 - RT-PCR: A Science and an Art Form

Abstract

RT-PCR is that technology by which RNA molecules are converted into their complementary DNA (cDNA) sequences by reverse transcriptase, followed by the amplification of the newly synthesized cDNA by standard PCR procedures. This approach to study gene expression is universally known as RT-PCR, because of the role of reverse transcriptase (RT) in the synthesis of first-strand cDNA. RT-PCR is a two-step process. It involves reverse transcription of purified RNA by RT via an appropriate method for priming and amplification of first-strand cDNA using some variant of PCR. Normalization of samples is very important in RT-PCR, and the efficiency of first-strand cDNA synthesis is one of the most important determinants in 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 useful primers which promote a balance between template specificity, thermodynamic stability when base-paired to the template, and 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.