Total RNA Extraction, Reverse Transcription, and Real-Time Polymerase Chain Reaction

RNA extraction and DNAse treatment were performed as previously described (Alboni et al., 2013a) using GenElute™ Mammalian Total RNA Miniprep Kit and DNase70-On-Column DNAse I Digestion Set (Sigma-Aldrich®, Milan, Italy). Two µg of total RNA was reverse-transcribed with High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, United States) and Real-Time PCR was performed, as previously described (Benatti et al., 2011), in ABI PRISM 7900 HT (Thermo Fisher Scientific, Waltham, MA, United States) using Power SYBR Green mix (Thermo Fisher Scientific, Waltham, MA, United States) and specific forward and reverse primers at a final concentration of 150 nM (see Supplementary Table S1 for primer sequences). Ct (cycle threshold) value was determined by the SDS software 2.2.2 (Thermo Fisher Scientific, Waltham, MA, United States); mRNA expression was calculated with the ΔΔCt method with cyclophilin A (CypA) as endogenous control.

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