FAQs

20 kb RNA reverse transcription can be achieved with all our RTases because RTases jump on and off their primed substrates. Secondary structures, unspecific binding and mis-priming can be the most common causes for the reaction ending prematurely.

We advise using specific primers or oligo(dT) and optimising each aspect of the reaction, for example, the different enzyme concentrations, primers, primer concentration, temperature and temperature cycling and time. Also, the subsequent PCR/qPCR step might need optimisation.

We recommend UltraScript™ 2.0 RTase for reverse transcribing long templates because it can sustain higher temperatures for longer, which may be necessary as the probability of secondary structures in RNA increases with its length.

We’ve included the following paper for your reference¹.

1  Thiel, V. et al. Effective amplification of 20-kb DNA by reverse transcription PCR. Anal Biochem 252, 62-70, doi:10.1006/abio.1997.2307 (1997).