Cfx fluorescent quantitative pcr instrument

Real-time PCR amplification was performed using an CFX fluorescent quantitative PCR instrument (Bio-Rad Laboratories, Hercules, CA, USA) in a total volume of 20 μL. The primer sequences are shown in Table S1. Actin was used as a reference gene. The PCR reaction mixture contained 4 μL 5× reaction buffer, 0.5 μL oligo (dT)18 primer (100 μM), 0.5 μL random hexamer primer (100 μM), 1 μL servicebio RT enzyme mix, and 110 μL total RNA. PCR amplification was performed using the initial heating for denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s. Then, the melting curve was analyzed at the end of every program [25 (link)]. Each PCR reaction was performed in triplicate. The 2^−ΔΔCT method was used to calculate each gene’s relative expression level [26 (link)].