Chapter 18 - 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 transcriptases, 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. Designing useful primers necessitates promoting a proper 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. The probable collaborative behavior of one or more pairs of oligonucleotide primers is best described in terms of the Tm of each primer involved. The Tm is that temperature at which 50% of the possible annealing events between primer and template have occurred and 50% have yet to occur.