Quantitative real-time PCR (qRT-PCR) analysis
Total RNA was extracted from the samples using EZ-10 Total RNA
Mini-Preps Kit (Sangon, Shanghai, China) according to the standard
manufacturer’s instructions. For qRT-PCR analysis, ChamQ SYBR qPCR
Master Mix (Vazyme, Nanjing, China) was used and PCR amplification were
performed following the instruction. Gene expression analysis using the
2–ΔΔCT method in CFX Manager™ Software v3.1 in CFX
Connect Real-Time PCR Detection System (Bio-Rad, California, USA). Three
replicates were run for each sample. Actin was used as an
internal control for normalization.