*2.7. RNA Isolation and RT-qPCR*

Total RNA was extracted by using Trisure Reagent (Bioline). RNA quality was assessed by A260/A280 ratio in a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). Briefly, RNA (1 μg) was subjected to reverse transcription (iScript, Bio-Rad, Madrid, Spain). An amount of 10 ng of the resulting cDNA was used as a template for real-time PCR amplifications. The mRNA levels for specific genes were determined in a CFX96 system (Bio-Rad). For each PCR reaction, cDNA template was added to Brilliant SYBR green QPCR Supermix (Bio-Rad) containing the primer pairs for either gene or for glyceraldehyde 3-phosphate dehydrogenase (*GAPDH*) and hypoxanthine phosphoribosyltransferase (*HPRT*) as housekeeping genes (Supplementary Materials Table S2). All amplification reactions were performed in triplicate and average threshold cycle (Ct) numbers of the triplicates were used to calculate the relative mRNA expression of candidate genes. The magnitude of change of mRNA expression for candidate genes was calculated by using the standard 2−(ΔΔCt) method. All data were normalized to endogenous reference (*GAPDH* and *HPRT*) gene content and expressed as relative fold-change of control.
