qPCR Analysis of Gene Expression

Total RNA samples were extracted with the RNAiso plus reagent (Takara, Japan). After digestion with gDNA digester (Yeasen, China), cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Yeasen). The reference gene RP18 was chosen as internal control (Pfaffl et al., 2004 (link); Huggett et al., 2005 (link)). The reaction mixture consisted of 5 µl TB Green Premix Ex Taq II (Tli RNaseH Plus), 1 µl of cDNA, and 0.25 µl of forward and reverse primers (see Supplementary Table S2; 10 µM) in a final reaction volume of 10 µl. The qPCR protocol included an initial denaturation step at 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s and 72 °C for 30 s. The relative expression of β-Actin was calculated by the $2^{-\mathrm{\Delta }\mathrm{\Delta }{C}_{\mathrm{T}}}$ method (Livak and Schmittgen, 2001 ; Shi et al., 2013 (link)). All experiments were repeated in triplicate.