Reverse Transcription–Polymerase Chain Reaction

Abstract

Reverse transcription coupled to the polymerase chain reaction (RT–PCR) is commonly used to detect the presence of mRNAs, pre-mRNAs, or other types of RNA such as noncoding RNAs. The method involves using a primer annealed to the RNA of interest. For mRNA, the primer is usually a synthetic oligo(dT)15–18, a random hexamer mixture (dN)6, or a synthetic DNA oligonucleotide that is complementary to a specific transcript (a gene-specific primer). This DNA:RNA hybrid serves as a template during reverse transcription, in which the enzyme reverse transcriptase (RT) generates a single-stranded cDNA copy of a portion of the target RNA molecule. Using random hexamer priming, it is possible to obtain representative cDNA copies of sequences from the entire length of the mRNAs and pre-mRNAs in a population. This cDNA can then be used as a template for PCR. On addition of gene-specific primers, a specific DNA fragment corresponding to a portion of the RNA of interest is generated.