RNA Extraction and cDNA Synthesis Protocol

Adult tissues or whole embryos were homogenized in Tri Reagent (Sigma) and total RNA was extracted as previously described [37 (link)] and treated with DNase I (Roche, Indianapolis, IN). An aliquot of each extract was used for spectrophotometry to determine RNA quality and concentration. RNA with a 260/280 ratio between 1.95–2.2 and a 260/230 ratio > 1 and < 3 was considered satisfactory and was used in this study. Each RNA extract was assayed in triplicate and an average value was determined. A 1 μg aliquot was taken of each sample and electrophoresed on an agarose gel to confirm quality and concentration. cDNA was synthesized from total RNA (5 μg; 20 μl final reaction volume) with oligo(dT) priming using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For analysis of 18s, reverse transcription was carried out using random primers which results in a lower reaction efficiency. A minimum of two RT reactions were performed for each biological replicate for technical replicate comparison.