2.5. Real-Time Quantitative PCR

We used the Trizol method to extract RNA, and cDNA was obtained by conducting reverse transcription of 3 μg of RNA according to the GeneCopopoeia (Rockville, MD, US) reverse transcription kit manual (Cat: AORT-0020). The cDNA was diluted three times, and the SYBR Green system was used for real-time quantitative detection. The final volume was 20 μL, with a total of 40 cycles. Settings were as follows: predenaturation at 95°C for 10 min, denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 15 s, and the heating rate from 72°C to 95°C was 0.5°C/6 s. All data were collected by the software of the Bio-Rad CFX96 PCR system. The collected Ct values were calibrated with GAPDH as an internal reference. The target gene expression was analyzed using the ^2−∆CT method [31]. Primers and sequences are listed in Supplementary Table 2.