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  • FAQ: How can I detect/amplify RNA targets?

    Isothermal amplification methods can typically be used for RNA targets with one-step reverse transcription reactions. Addition of AMV (NEB #M0277) or MMuLV (NEB #M0253) Reverse Transcriptases is compatible with the buffer and temperature used for many isothermal amplification reactions, simplifying the reaction workflow. A separate cDNA synthesis step can first be performed, if desired, but for most detection reactions speed and simplicity are preferred. In some cases, the DNA polymerase itself can prove sufficient for single-enzyme RT isothermal amplification. Bst DNA Polymerase, Large Fragment has been observed to have a low level of RT activity, particularly in the high magnesium concentrations of isothermal amplification reactions (~8 mM), but it is highly variable and cannot match the performance of two-enzyme mixes. However, Bst 2.0 has significantly higher reverse transcriptase activity and can perform RT-isothermal amplification without a second enzyme, in many cases. While a two-enzyme mixture will prove most consistent and robust, we recommend Bst 2.0 for situations where a single enzyme is preferred.