The micro-punched samples were immediately homogenized in Trizol reagent (Invitrogen, Carlsbad, CA) and processed according to the manufacturer’s protocol, but with twice the suggested ratio of Trizol to tissue (Edenberg et al., 2005). Ethanol-precipitated RNA was further purified through RNeasy columns (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The yield, concentration, and purity of the RNA were determined by running a spectrum from 210 to 350 nm, and analyzing the ratio of large and small ribosomal RNA bands using an Agilent Bioanalyzer. Yields, purity, and quality of the RNA were excellent; RNA integrity numbers (RIN) averaged 8.5 for the samples, showing little or no degradation.
