RNA Isolation, Purification, and cDNA Synthesis

Total RNA was isolated from 100 mg frozen tissue using 1 mL TRIzol ® reagent (ThermoFisher Scientific, Waltham, MA USA) according to the manufacturer’s instructions. The RNA was treated with DNase I (ThermoFisher Scientific) at 37 ° C for 25 – 30 min and then purified by phenol/chloroform extraction and stored at -80 ° C. RNA quantity and quality were determined by spectrophotometer readings (NanoDrop, ThermoFisher Scientific) and RNA integrity numbers (RINs) obtained from an Agilent 2100 bioanalyser [29 (link)]. Genomic DNA elimination was confirmed by PCR using primers specific for genomic DNA. First strand cDNA was synthesized from 2 μg RNA using 100 units M-MLV-RT (ThermoFisher Scientific) with 2.5 μM random decamers and 2.5 μM oligo dT primers (ThermoFisher Scientific), 0.5 mM of each dNTP and 20 units RNase inhibitor in RT Buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2 and 5 mM DTT) in a final volume of 20 μL. The reaction was incubated at 44 ° C for 1 h then inactivated at 92 ° C and the product aliquoted and stored at -20 ° C.