Quantitative Gene Expression Analysis

The mRNA levels of selected genes were assessed by quantitative real-time PCR (qRT-PCR) as we previously described [27 (link)]. Briefly, RNA was isolated from tissue samples using a NucleoSpin RNA Plus Kit with RNase-free DNase I treatment (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) and an EvoScript universal cDNA master kit (Roche Molecular Systems, Boston, MA, USA) was used to synthesize cDNA. Next, qRT-PCR was carried out using the LightCycler 96 System with FastStart essential DNA green master (Roche Molecular Systems, Rotkreuz, Switzerland) together with the verification of PCR product specificity by melting curve analysis. Primers sequences are listed in Supplementary Table S1. The mRNA levels of target genes were normalized to β-actin and calculated according to the Pfaffl method [28 (link)]. All samples were assayed in duplicate.