Reverse transcription PCR


Reverse transcription PCR (also RT-PCR) is a method used to amplify small quantities of RNA via a cDNA intermediate.

It involves annealing an anti-sense gene specific primer to mRNA, which is extended using reverse transcriptase to make single-stranded cDNA. Then another (sense) primer is annealed to the nascent cDNA and extended to make double-stranded cDNA. Once cDNA is double-stranded, it can be denatured, have primers annealed to both strands and extended again, in a PCR fashion. Furthermore, different primers can be used to identify different mRNA splice variants.

RT-PCR is not useful in determining the size of transcripts, and there is a difficulty in that its sensitivity can lead to the amplification of contaminants.