Quantitative Real-Time PCR Protocol

For qRT-PCR analyses, gene-specific oligonucleotide primers were designed and described in Table 3. The gene specificity of each pair of primers was checked by melting curves and product re-sequencing twice. The GAPDH gene was employed as the internal control for calculating relative expression of the mRNA [53 (link)]. The sequences of GAPDH primers are described in Table 3. Real-time PCR was performed using FastStart Universal SYBR Green (Roche, Basel, Switzerland), initiated by 10 min at 95 °C and followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and then by 72 °C for 10 min, and completed with a melting curve analysis program. The PCR mixture (10 μL total volume) was comprised of 5 μL of Roche FastStart Universal SYBR Green Master (ROX) (Roche, Basel, Switzerland), 0.75 μL of each primer (10 μM), 0.5 μL of diluted cDNA and 3 μL of PCR-grade ddH₂O. No-template controls and melting curve analysis were included for each gene during each run.