Gene expression analysis by quantitative PCR (qPCR)

Total RNA extracts were prepared using PureLink® RNA Mini Kit and DNase set (Invitrogen) according to manufacturer’s protocol. 25 ng RNA was used as template in 20 μL reactions for qPCR using EXPRESS One-Step SYBR GreenER kit (Thermo Fisher Scientific), with ROX as reference dye, and 200 nM each of forward and reverse primers for genes of interest. No template and no reverse transcriptase controls were included and samples were analyzed in duplicate. Reactions were run using the QuantStudio 6 Flex Real-Time PCR System (Applied Biosciences) with the following conditions: (a) 5 min at 50°C and 2 min at 95°C (cDNA synthesis), (b) 15 s at 95°C and 1 min at 60°C (40-cycle amplification), and (c) melt curve analysis. Gene expression was expressed as dC_T or fold-change (FC) relative to WT, calculated based on the 2^−ΔΔC_T method, with GAPDH as the reference gene.