Total RNA was isolated using TRIzol (TransGene Biotech, China) and reverse-transcribed into cDNA using a Reverse Transcription Kit (Takara, Dalian, China). Real-time PCR (RT-PCR) was performed using an ABI Prism 7500 sequence detection system (Applied Biosystems, CA, USA). Each reaction (20 μL) contained 2 μL of cDNA template, 10 μL of SYBR Green Master Mix (Takara, Dalian, China), 0.4 μL of ROX Reference Dye II, 0.8 μL of each of the forward and reverse primers, and 6 μL of RNase-free ddH2O. Supplementary Table 1 shows the primer sequences used for RT-PCR. The reactions were performed in a 96-well plate using the following incubation protocol: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. The expression of the genes was normalized to the expression of β-actin by comparing the cycle threshold (Ct) values. Gene expression was determined by the 2−ΔΔ Ct method and then analyzed for statistical significance.

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