qRT-PCR Analysis of Gene Expression

For qRT-PCR analyses, gene-specific oligonucleotide primers were designed (Table 2) and the gene specificity of each pair of primers was checked by melting curves and product re-sequencing twice. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was employed as the internal control for calculating relative expression of the mRNA [56 (link)]. The sequences of GAPDH primers are described in Table 1. qRT-PCR was performed by FastStart Universal SYBR Green (Roche Diagnostics, Shanghai, China), initiated by 10 min at 95 °C and followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 10 min, and completed with a melting curve analysis program. The PCR mixture (10 μL total volume) comprised 5 μL of Roche FastStart Universal SYBR Green Master (ROX), 0.75 μL of each primer (10 μmol/L), 0.5 μL of diluted cDNA and 3 μL PCR-grade ddH₂O. No-template controls and melting curve analysis were included for each gene during each run.