Complementary DNA (cDNA) synthesis by reverse transcription

For rotavirus and norovirus, cDNA was synthesized using Promega cDNA synthesis kit (Promega, Madison, USA). For reverse transcription (RT), 3 μL of extracted RNA (100 ng) was mixed with a reaction mixture, consisting of 1 μL of oligo dT primer (25 μg/ml) and 1 μL of nuclease free water in nanocentrifuge tube, kept at 70°C for 5 min and chilled for 5 min in ice. After that 4 μL of 5X reaction buffer, 2 μL of 5 mM MgCl₂, 1 μL of 10 mM PCR Nucleotide Mix, 0.5 μL of ribonuclease inhibitor (1 u/ μL), 1 μL of reverse transcriptase, and 6.5 μL of nuclease free water were mixed for each sample in the same nanocentrifuge tube, containing 5 μL of previously chilled genomic RNA. Total volume of the mixture was 20 μL. The mixture was heated at 25°C for 5 min, 42°C for 60 min and 70°C for 15 min. The cDNA was prepared and stored at -20°C.