What are the reaction components for reverse transcription?
Posted March 1, 2023
Enzyme Reverse Transcriptase - Reverse transcriptase is a multifunctional enzyme that has RNase H (ribonuclease H) activity as well as RNA-dependent and DNA-dependent DNA polymerase activity. It is responsible for catalyzing the reverse transcription reaction and transcribing double-stranded DNA from single-stranded RNA.
Primers - Primers are short DNA oligonucleotides that serve as a starting point for synthesizing a new strand by binding to complementary sequences on the RNA template. Reverse transcriptase requires primers to initiate reverse transcription.
RNA template - The RNA template used for reverse transcription must be pre-treated to remove genomic DNA.
Reaction Buffer - The reaction buffer helps to maintain a favorable pH and ionic strength, ensuring proficient reverse transcription. It may also contain additives that help to boost the efficiency of the reaction.
Deoxynucleoside Triphosphates (dNTPs) - Freshly diluted, high quality (dNTPs) at 0.5 -1 mM each, preferably at equimolar concentrations enhance the efficiency of reverse transcription.
Dithiothreitol (DTT) - DTT is a reducing agent that helps to optimize enzyme activity. It must be dissolved and mixed well with other reaction components to prevent precipitation, which may compromise the efficiency of the reaction.
RNase Inhibitors - RNase inhibitors help prevent RNA degradation during reverse transcription. The appropriate RNase inhibitors will depend on their mode of actions and the reaction requirements. They may be included in the reaction buffer or added directly to the reaction.
RNase-Free Water - Using nuclease-free water in reverse transcription is recommended. Simple filtration is not effective at removing contaminating RNases. Moreover, RNases are heat stable, making autoclaved water inadequate for use in the reaction.