Each 20 µL of reaction mixture contained 10 µL of SYBR Green Mix, 6 µL of nuclease-free H2O, 2 µL of cDNA, 1 µL of upstream primer, and 1 µL of downstream primer. The qPCR thermal cycling conditions were as follows: Initial denaturation at 95 °C for 60 seconds, followed by 40 cycles of 95 °C for 15 seconds, 60 °C for 15 seconds, and extension at 72 °C for 45 seconds. The primers were synthesized by Tsingke, Ltd. Relative mRNA expression levels were calculated using the 2-ΔΔCq method, with β-actin used as an internal reference gene.
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