RNA Isolation and qPCR Gene Expression Analysis

RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer’s protocol as previously described^{1 (link)}. All procedures were carried out using RNase-free reagents and consumables. One microgram of RNA was reverse transcribed into cDNA using Moloney-murine leukemia virus (M-MLV) reverse transcriptase and random primers (Promega, Madison, Wisconsin, USA) in 20 μl reaction volume. Quantitative PCR (qPCR) assays were carried out using a Mastercycler ep realplex S (Eppendorf, Sydney, Australia) and the fluorescent dye SYBR Green (Bio-Rad), following the manufacturer’s protocol. Briefly, each reaction (20 μl) contained 1 × mastermix, 5 pmol of primers and 1 μl of cDNA template. Amplification was carried out with 40 cycles of 94 °C for 15 s and 60 °C for 1 min. Gene expression was normalized to the geometric mean of three housekeeper genes, GAPDH, β-actin and PPIA. A no-template control was included for each PCR amplification assay. The level of expression for each gene was calculated using the comparative threshold cycle (Ct) value method using the formula 2^−ΔΔCt (where ΔΔCt = ΔCt sample—ΔCt reference).