Quantitative PCR analysis
Quantitative PCR (qPCR) was performed as previously described
(Jin et al., 2020). Briefly, total RNA
was extracted using TRizol (Life Technologies, Frederick, MD, USA),
followed by ethanol extraction. qPCR was conducted in triplicate using
SYBR Green PCR reagent (Bio-Rad, Hercules, CA, USA) on the CFX96
Real-Time PCR system (Bio-Rad). PCR conditions included a denaturation
step of 95°C for 5 min, followed by 40 cycles consisting for 95°C for 15
sec, 60°C for 30 sec, and 72°C for 30 sec. The primer sequences are
described in Supplementary Table 2. The relative expression of each
target gene was calculated by the 2-ΔΔCt method after
normalization to β-Actin.