Quantitative Gene Expression Analysis

RNA extraction and reverse transcription were carried out as previously described (22 (link)). RNA quality and quantity were assessed by spectrophotometry (NanoDrop ND-1000; Thermo Fisher Scientific, Waltham, MA, United States) and its integrity was determined with an Agilent 2,100 Bioanalyzer (Agilent Technologies Inc., Waldbronn, Germany). In all cases, the RNA integrity was ≥9 and the A260/280 ratio was between 1.96 and 2.02. Total RNA was reverse-transcribed using an iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, United States). For real-time PCR determinations, we used a cDNA template in a 20-μL reaction solution containing 0.2 μmol/L of each primer and SsoAdvanced™ Universal SYBR^® Green Supermix (Bio-Rad). The primers used are listed in Table 2. Real-time PCR was performed on a MiniOpticon Real-Time PCR System (Bio-Rad). Each PCR run included duplicates of reverse transcript cDNA for each sample and negative controls (reverse transcription-free samples, RNA-free sample). Quantification of the target gene transcripts was conducted using glucuronidase beta (Gusβ) gene expression as reference and was performed with the 2^−ΔΔCT method (23 (link)). Product fidelity was confirmed by melting curve analysis.