Quantification of Gene Expression via qRT-PCR

Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer’s instructions. Then, RNA was transformed to cDNA with reverse transcription using a PrimeScript II RT Reagent Kit (Takara, Kyoto, Japan). Quantification of specific gene expression was measured with SYBR premix Ex Taq™ (Takara) and a Prism 7900HT sequence detection system (Thermo Fisher Scientific, Rockford, IL, USA) following the manufacturer’s protocols. Relative gene expression was determined using the 2^ΔCt (internal control) − ΔCt (gene) method and normalized to cyclophilin [53 (link)]. Data was acquired from at least triplicate experiments and denoted as mean ± standard deviation (SD). Used primer sequences are listed in Supplementary Table S2.