Higher incubation temperatures can help eliminate problems of template secondary structure. In addition, high temperature improves the specificity of reverse transcription by decreasing false priming. Thus, thermoactive reverse transcriptases that can be incubated at high temperatures (5070C) are more likely to produce accurate copies of mRNA, especially if the template has a high GC content.
Note: At these high temperatures, use only specific primers; do not use oligo(dT) or random hexamer primers.RNase H activity
In contrast to Tth DNA Polymerase, AMV and M-MuLV Reverse Transcriptases
have RNase H activity. This activity will specifically degrade RNA in
an RNA:cDNA hybrid. This can be detrimental if degradation of template
RNA competes with DNA synthesis during production of first-strand cDNA.
M-MuLV Reverse Transcriptase has lower RNase H activity than AMV Reverse
Transcriptase. Mutated enzymes without RNase H activity are also available,
Reverse Transcriptase (Cat. No. 1785826) or the C.
therm. Polymerase for Reverse Transcription in Two-Step PCR (Cat.