2.10. Real-Time Quantitative PCR

Four treatments and the RNA extraction were carried out as above. About 1 μg total RNA was used to erase gDNA at 42°C for 2 min and then reversely transcripted into cDNA using the TB GREEN kit (TaKaRa Bio Inc., Dalian, China). Real-time PCR was performed with the CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in a 20 μL PCR reaction system containing 2 μL cDNA, 1 μL reverse primer,1 μL forward primer, 10 μL SYBR mixture, and 6 μL deionized water. The GAPDH was used as a housekeeping gene to normalize the gene levels, and the RT-PCR data were analyzed using the 2^-ΔΔCT method (Livak and Schmittgen, 2001). All the primers used in this study were synthesized by Shanghai Shenggong Biotech Co., Ltd., Shanghai, China (Supplementary Table 1).