RT-PCR Protocol for Gene Expression Analysis

Primers were chosen based on the results from previously published papers [15 (link),16 (link)] and listed in Table 2. RT-PCR was conducted by a LightCycler480 system (Roche, Castle Hill, NSW, Australia) using the TB Green^® Premix EX Taq^TM kit (TaKaRa, Dalian, China). The RT-PCR parameters were as follows: 95 °C for 30 s, then 40 cycles of denaturation (95 °C, 5 s), annealing (60 °C, 20 s), and extension (72 °C, 1 min). The specificity of the PCR reaction was confirmed through a melting curve analysis. The efficiency of PCR amplification for each gene was checked with the dilutions of the samples. The GAPDH was used to normalize the relative expression levels of target genes [16 (link)]. The 2^−ΔΔCt method was used for data analysis, as previously described [17 (link),18 (link)].