( ...)Choice of RT-PCR Enzymes

Obviously, in RT-PCR a major factor to consider is the choice of reverse transcriptase used to synthesize cDNA. Since each of the available enzymes has different enzymatic properties, one may be more suitable for a specific experiment than the others. Among the enzyme properties to consider are: Temperature optima

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, e.g. Expand Reverse Transcriptase (Cat. No. 1785826) or the C. therm. Polymerase for Reverse Transcription in Two-Step PCR (Cat. No. 2016311)
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