Reverse Transcriptase PCR (RT-PCR)

RT-PCR is an extremely useful variation of the standard PCR which permits the amplification of specific mRNA transcripts from very small biological samples without the need for the rigorous extraction procedures associated with mRNA purification for conventional cloning purposes. Conveniently, the dNTPs, buffer, Taq polymerase, oligonucleotide primers, reverse transcriptase (RT) and the RNA template are added together to the reaction tube. The reaction is heated to 37 °C which allows the RT to work and permits the production of a cDNA copy of the RNA strands that anneal to one of the primers in the mix. Following ‘first strand synthesis’, a normal PCR is carried out to amplify the cDNA product, resulting in ‘second strand synthesis’, and subsequently a dsDNA product is amplified as usual. The choice of primer for the first strand synthesis depends on the experiment. If amplification of all mRNAs in the cell extract is required, then an oligo dT primer that would anneal to all the polyA tails can be used. If a specific cDNA is sought, then a coding region-specific primer can be used with success, otherwise a random primer could be used. The method is fast, accurate and simple to perform. It has many applications, such as the assessment of transcript levels in different cells and tissues . When combined with allele-specific primers, it also allows the amplification of cDNA from single chromosomes. RT-PCR is widely used as a diagnostic tool in microbiology and virology.