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.
RT-PCR Process
- After RNA is released from cellular material through extraction, an aliquot of the extracted sample is added to a reaction mixture that 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 95°C, and the RNA/DNA strands are denatured.
- The temperature is 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 in one or two steps. 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, the RNA to cDNA is first transcribed. Transcription occurs between 40°C and 50°C, depending on the properties of the reverse transcriptase enzyme utilized; products of that reaction are then amplified in a separate response.
