RT (reverse transcribed)-PCR

In a number of situations, when mRNA transcript needs to be examined, reverse transcription is" first used, to obtain cDNA, which is then used as a template for PCR reaction. For this purpose, mRNA is first isolated, purified and subjected to reverse transcription with the help of reverse transcriptase enzyme. Often the synthesis of cDNA is initiated by primers that are either complementary to poly A tail (for mRNA) or complementary to specific sequences known to be present on one or more specific subsets of RNA species.

Adapters of known sequences may also be ligated to mRNA to facilitate designing of primers. cDNA synthesized thus, by RT-PCR, is then used as a template for PCR, using Taq polymerase and specific primers. One of the important uses of RT-PCR is the identification of differences in two mRNA samples, using the technique of differential display, leading to isolation of specific genes.