Quantifying Gene Expression by RT-qPCR

We followed the methods of Wang et al. [24 (link), 25 (link)]. Total RNA was extracted with TRIzol and assessed by spectrophotometer. Then, reverse transcription of RNA from each group was performed using Prime Script RT reagent Kit (Beyotime Biotechnology, Shanghai, China). Primer was designed and synthesized by Shanghai Biotechnology Service Company in accordance with Gene sequence in GenBank Gene sequence design, together with Oligo v6.6 (sequences as Table 1). qPCR was performed using Premix Ex Taq SYBR-Green PCR (Takara) according to the manufacturer's instructions on an ABI PRISM 7300 (Applied Biosystems, Foster City, CA, USA). The mRNA level of individual genes was normalized to GAPDH and calculated by 2^−ΔΔCTdata analysis method.