Quantitative RT-PCR Analysis of Gene Expression

Total RNA from tested tissues was extracted by RNAiso (TaKaRa). According to the manufacturer’s instructions, the first-strand complementary DNA (cDNA) synthesis was carried out with 1 μg total RNA by the PrimeScript™ RT reagent Kit (TaKaRa). The qRT-PCR was performed on the ABI 7500 real-time PCR machine with a total 20 μL reaction, which contained SYBR (TaKaRa) 10 μL, 10 mM forward and reverse primer 0.4 μL respectively, and diluted cDNA 0.1 μL. The ACTIN gene was adopted as the internal control for normalization (Cao et al., 2018 (link)). The qRT-PCR results were obtained from three biological replicates. The relative expression levels were summaried based on the 2−ΔΔct method (Livak and Schmittgen, 2001 (link)).