RNA Extraction and qPCR Analysis

Total RNA was isolated using TRIzol reagent (ThermoFisher, Illkirch, France), as described by the manufacturer. Reverse transcription was performed with 1µg of total RNA using random primers and with M−MLV enzyme (ThermoFisher, Illkirch, France). Real time quantitative PCR was performed with SYBR green Master Mix (Roche, Meylan, France), on a Light Cycler 480 instrument (Roche) as previously described [22 (link)]. Ribosomal protein S9 (rS9) and GAPDH were used as an internal control. The sequence of the primers used in this study is indicated in Table S1. Results are expressed as N−fold differences in target gene expression relative to the internal control gene and termed “mRNA expression”, determined as mRNA expression = 2 − Δ Ctsample, where the Δ Ct value of the sample was determined by subtracting the Ct value of the target gene from the Ct value of the average of the internal control genes. Target genes were considered to be non−detectable when the Ct value was above 35.