2.8. Detection of mRNA Levels by q-PCR

RNA was extracted using TRlzol, according to the manufacturer's instructions (CWBIO, Beijing, China). The extracted RNA was reverse transcribed to produce cDNA using a HiFiScript cDNA Synthesis Kit (CWBIO, Beijing, China). cDNA was then further amplified and detected using q-PCR with the UltraSYBR Mixture (CWBIO, Beijing, China), according to the manufacturer's instructions, and a CFX96 thermocycler (Bio-Rad, California, USA). The q-PCR parameters were predenaturation at 95°C for 10 min, denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 32 s. Samples were amplified over 45 cycles of denaturation, annealing, and extension. The relative quantitative 2^-△△Ct method was used for analysis, where △Ct = Ct (target gene) − Ct (internal reference gene) and △△Ct is obtained by subtraction of △Ct of the study sample from △Ct of the control sample. Fold change was determined using 2^-△△Ct.