Reverse Transcription of the Ribonucleic Acid: The First Step in RT-PCR Assay

Reverse transcription (RT) is the synthesis of complementary deoxyribonucleic acids (DNA) from single-stranded ribonucleic acid (RNA) templates. This process is catalyzed by the reverse transcriptase enzyme, which is the replicating enzyme of retroviruses. Reverse transcriptase was discovered in 1970, and since then, it has played an instrumental role in the advancement of molecular biology and biotechnology research. In the presence of all four deoxynucleotides (dNTP: dATP, dCTP, dGTP, and dTTP) and under well-defined salt and pH conditions, the reverse transcriptase extends a primer complementary to the RNA to produce a complementary DNA (cDNA) for the RNA template. In this chapter, a simple method of reverse transcription of total cellular RNA into cDNA is described using Superscript II reverse transcriptase (Invitrogen); the resulting cDNA can be used in polymerase chain reaction (PCR).