Reverse transcriptase PCR (RT-PCR) was developed to amplify RNA targets (RNA viruses such as HIV, HCV, and influenza are key examples). Essentially, the method entails an initial step of transcribing a portion of the RNA genome into complementary DNA (cDNA) which is then amplified through PCR.
PCR depends on the Taq Polymerase enzyme; RNA is not an efficient substrate for this enzyme. This is why the target of interest (if present) is first transcribed into complementary DNA (cDNA), which can then be amplified.
- After RNA is released from cellular material through extraction, an aliquot of the extracted sample is added to a reaction mixture which contains reverse transcriptase enzyme, primers specific for the target of interest, and nucleotides.
- If the target is present, primers anneal to the RNA strand.
- Reverse transcriptase enzyme synthesizes a complementary DNA strand, extending from the primer.
- The temperature is raised to 95o C, and the RNA/DNA strands are denatured.
- The temperatures are lowered, allowing primers to anneal to the newly formed cDNA.
- Polymerase enzyme synthesizes a new DNA strand, extending from the primer.
- Multiple cycles geometrically increase the number of copies of DNA.
RT-PCR can be performed as one or two step procedures. In a one-step procedure, the reverse transcriptase is performed in the same reaction tube as the polymerase chain reaction. In a two-step procedure, transcription of the RNA to cDNA is performed first. Transcription occurs between 40o C and 50o C, depending on the properties of the reverse transcriptase enzyme utilized; products of that reaction are then amplified in a separate reaction.