Quantitative Analysis of mRNA Expression

Total RNA of the VG and CA were separately extracted. cDNA was synthesized from 1 μg RNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China). All specific primers for qPCR were designed by AlleleID 6 software (PREMIER Biosoft, Palo Alto, CA, United States). The qPCR was run in the CFX96^TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States) using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) according to the manufacturer’s protocol. The programs were set as follows: enzyme activation at 95°C for 30 s, followed by 40 cycles with denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, and a melting curve analysis. mRNA expression levels were normalized to the reference (28S rRNA) (Ballinger and Perlman, 2017 (link)), and quantified based on the comparative 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)). The experiments were repeated 3 times.