To extract the RNA, punches were immediately homogenized in Trizol (Life Technologies, Carlsbad, CA) and processed according to manufacturer’s protocol but with twice the suggested ratio of Trizol to tissue (Edenberg et al., 2005). Ethanol-precipitated total RNA was further purified through Qiagen RNeasy columns (Qiagen, Hilden Germany) according to the manufacturer’s protocol. The yield, concentration and purity of the RNA were measured by Nanodrop (Thermo Fisher Scientific, Waltham, MA) spectrum from 220 nm to 340 nm. Quality was also assessed by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Ca); RNA integrity numbers (RIN) averaged 8.6 for the samples, showing little degradation.
