Spss statistical software package 22

qRT-PCR was performed in a 20 μl reaction volume using the ABI 7300 real-time PCR system (Applied Biosystems, USA) with the SYBR Premix Ex Taq (Takara, Japan) according to manufacturer’s protocol. The reaction volume contained 1 μl (approximately 100 ng) of cDNA, 1 μl of each sense and anti-sense primer (10μM), 10 μl of 2 × SYBR Green Premix and 7 μl of ddH₂O. The following PCR running program was used: 95 °C for 60 s, followed by 40 cycles of 95 °C for 10 s, 57 °C for 30 s and 72 °C for 30 s. The PCR reaction for each of three biological replicates was implemented according to the procedure described above, and each reaction was performed in triplicate. Melting curves were used to analyze the specificity of amplifications. Specific primers for all the genes used in this study were designed using Beacon Designer 7 (Supplementary Table 11), and the ribosomal protein S20 gene (S20) was used as the endogenous control [52 (link)]. The relative expression level of each gene was analyzed according to the 2^-ΔΔCTmethod [55 (link)]. Data are presented as the means ± standard deviation (SD). Statistical analysis, one-way ANOVA, was performed using the IBM SPSS statistical software package 22.0, with significance declared at P < 0.05.