Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA extraction was conducted using Tissue Total RNA Isolation Kit V2 (RC112, Vazyme, China) according to the protocol. RNA concentration was measured using A260/A280 ratio and agarose gel electrophoresis. Total RNA (1000 ng) was individually reverse transcribed into cDNA using HiScript II Q RT Super Mix (R223-01, Vazyme, China). The reaction mix was incubated for 15 minutes at 50°C and 5 seconds at 85°C. RT‑qPCR was performed by ChamQ SYBR Color qPCR Master Mix (Q411, Vazyme, China). PCR was performed using a Real-Time PCR Quantification system (ABI 7500 fast; Applied Biosystems; Thermo Fisher Scientific, Inc.). The mRNA expressions were determined by analyzing data using the 2−ΔΔCT method, and GAPDH served as the control of mRNA.