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.