10.2.1. Thermostable reverse transcriptases

Thermostable reverse transcriptases, operating at temperatures up to 50-70°C, avoid much non-specific priming of the RNA through elevated reaction temperatures (Lanford et al., 1995; Laskus et al., 1998; Craggs et al., 2001; Horsington and Zhang, 2007; Carrière et al., 2007; Celle et al., 2008). Thermostable reverse transcriptases need to be inactivated thoroughly prior to the PCR step, otherwise the reverse transcriptase has access to primers for both strands (thus nullifying the strand-specificity). Another strategy is to inactivate the (viral) RNA by alkaline treatment or digestion with RNase H (McGuiness et al., 1994), thereby removing any target for reverse transcription during PCR.

When using thermostable reverse transcriptase, make sure that the virus-specific portion of the tagged cDNA primer has a theoretical Tm ~60oC, to ensure adequate priming at elevated temperatures.