Real-time quantitative polymerase chain reaction analysis

Total RNA was extracted from tissue or cells using a commercial RNAprep pure kit (#CW0581, CWbio, China) according to manufacturer’s instructions. The isolated RNA was reverse transcribed into cDNA at 42 °C for 50 min and 85 °C for 5 min using a HiFiScript cDNA Synthesis Kit (#CW2569M, CWbio, China). Quantitative polymerase chain reactions (PCR) were performed with the UltraSYBR Mixture (Low ROX) (#CW2601M, CWbio, China) in a Q-PCR instrument (Lightcycler 96, Roche, Switzerland). The PCR protocol was 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 56 for 30 s, and 72 for 32 s. The primers used were shown in Table 1. Fold-change of the target gene mRNA expression was calculated using the 2^−ΔΔCT method.