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 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.
