Amplification of cDNA Generated by Reverse Transcription of mRNA: Two-Step Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Reverse transcription-polymerase chain reaction (RT-PCR) is a powerful method to detect and synthesize cDNA copies of low-copy-number mRNAs. Two enzymes are used: reverse transcriptase to produce single-stranded cDNA copies, which are then used as templates in an amplification reaction catalyzed by a thermostable DNA polymerase. For this reason, the method is known as "two-step RT-PCR." This protocol describes the traditional method of RT-PCR in which the two synthetic reactions are performed separately and sequentially.