GoScript™ Reverse Transcription System

Thaw the GoScript reverse transcriptase, 5x reaction buffer, and PCR nucleotide mix on ice without using a heating block. Mix and briefly centrifuge each component before use.

Combine experimental RNA, oligo dT primer and/or random or gene-specific primer, and nuclease-free water in a low-retention tube to achieve a final volume of 5 microliters.

Heat the RNA mixture at 70°C for 5 minutes to denature it, then immediately chill in ice water for at least 5 minutes. Centrifuge briefly to collect condensate and keep on ice.

On ice, combine nuclease-free water, 4 microliters of 5x reaction buffer, magnesium chloride (1.5-5 mM), 1 microliter of PCR nucleotide mix, 20 units of RNasin inhibitor, and 1 microliter of GoScript reverse transcriptase to achieve 15 microliters per reaction.

Dispense 15 microliters of the reverse transcription master mix to each reaction tube on ice, then add 5 microliters of the denatured RNA and primer mixture for a final volume of 20 microliters.

Incubate the reaction mixture at 25°C for 5 minutes to anneal primers, then at 42°C for up to 1 hour to extend the cDNA synthesis.

Heat the cDNA reaction at 70°C for 15 minutes to inactivate the reverse transcriptase before proceeding with endpoint or real-time PCR analysis. Reactions can be stored at -20°C for long-term storage.