Quantitative Gene Expression Analysis

Total RNA was extracted using the NucleoSpin RNA Plus kit (TaKaRa Biotechnology [Dalian] Co., Ltd., Dalian, China) in accordance with the manufacturer's protocol. RNA was reverse-transcribed to complementary DNA (cDNA) using the PrimeScript RT Reagent Kit (TaKaRa Biotechnology [Dalian] Co., Ltd.). RT-PCR analysis was performed using SYBR Green Master Mixture reagent (Takara Bio, Inc., Kusatsu, Shiga, Japan) and an ABI 7500-Fast Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA). The cycling conditions for cDNA amplification are described elsewhere (Zheng et al., 2018 (link)). The fold change in relative gene expression was calculated using the 2^−∆∆Ct method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference. The primers used for RT-PCR are listed in Supplementary Table S1.