RNA isolation, cDNA synthesis, and quantitative reverse transcription PCR

Total RNA was extracted using a Total RNA kit (DP419, Tiangen, Shanghai, China), and reverse transcription was performed using an All-in-One cDNA Synthesis SuperMix (Biotool, Houston, Texas, USA) for TaqMan analysis. qRT-PCR was performed using a 2×SYBR Green qPCR Master Mix (High ROX) (Biotool) on an Applied Biosystems 7900 Fast Real-Time PCR System (Applied Biosystems, Stadt, CA, USA). Relative quantitation of mRNA expression was performed using the ΔΔCt method. Ct values were first normalized within the sample to the housekeeping gene β-actin before comparison across the normal group samples. The mouse primers were synthesized by Sangon Biotech Co, Ltd (Shanghai, China).