RNA cannot be used directly as a template for PCR or Real-Time PCR. The Taq polymerases employed during PCR amplification are DNA template dependent DNA polymerases. Hence, when working with RNA an additional step, Reverse Transcription (RT) is necessary to generate cDNA (complementary to RNA sequence).
Reverse transcriptase’s are enzymes isolated from viruses with a RNA genome. These viruses utilize reverse transcriptase’s to generate cDNA in the process to replicate and re-create more viral particles in the host they attack. Typically RT used for molecular biology applications have been obtained from Mouse Murine Leukemia virus (M-MLV), Avian Myeloblastosis Virus (AMV), or are derivatives of these enzymes. To note, reverse transcriptase enzyme is also found in retroviruses and use for integration of cDNA during integration into the host genome.
To set up the RT reaction, RNA of good quality (free of gDNA) is necessary. Use of DNase I enzymes such as Turbo DNA-free or DNase I should be employed. RNA is combined with dNTP’s and requisite buffers (SuperScript IV VILO Master Mix) and RT enzyme (at 50-55°C) to generate the cDNA. cDNA can be measured using spectrophotometer or a Qubit fluorimeter.
RT reaction can be classified as:
- One step RT-PCR : RT and PCR amplification in a single tube using SuperScript IV One-Step RT-PCR System
- Two step RT-PCR: Synthesis and purification of cDNA, which is used as template for PCR amplification step