MMLV-RT

For RNA with very high secondary structure we recommend using 55C MMLV-RT (MDX117), this reverse transcriptase can be used at temperatures up to 60°C which improves the cDNA yield from difficult RNA targets that require higher temperature to denature strong secondary structures.

Avoiding ribonuclease contamination is of primary concern since high quality template RNA is required. You can add RNase Inhibitor (MDX056), alternatively we also have RNase-Tolerant MMLV-RT (MDX043) which includes an RNase inhibitor.

If you are going to make cDNA, you will require a buffer that contains 0.2 mM each dATP, dCTP, dGTP & dTTP, 10 mM DTT (dithiothreitol), 25 mM KCl, 3.5 mM MgCl2 and 50 mM Tris–HCl (7.5). However if you want to do RT-qPCR, we recommend 1-Step qPCR Buffer, 4x (MDX034) or Lyo-Ready 1-Step RT-qPCR Buffer (MDX052).

The reverse transcriptase can be inactivated by adding a chelating agent such as EDTA or heating to 70°C or higher for 10 min.

Yes, to initiate reverse transcription, the MMVL-RT reverse transcriptase require a primer to bind to its complementary sequences on the RNA template and serve as a starting point for synthesis of a new strand. For general cDNA synthesis, random hexamer primers and oligo(dT) primers can be used either separately or mixed. For RT-PCR and RT-qPCR gene-specific primers should be used.

Unlike eukaryotic replicative polymerases, reverse transcriptase lack exonuclease activity and so is error prone. MMLV-based reverse transcriptase has been reported to have an error rate in the range of 1.1 × 10⁻⁴ to 4.8 × 10⁻⁴.