Gene Expression Analysis from Total RNA

Total RNA from tissues and cells was isolated using the miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). Its concentration was assessed by NanoDrop 2000 (Thermo Fisher; Wilmington, DE, USA). Total RNA samples were reverse transcribed using TransScript First-Strand cDNA Synthesis SuperMix (TransGen; Beijing, China). Real-time PCR reactions were performed using SYBR Green qPCR SuperMix (Applied Biosystems Life Technologies; Foster, CA, USA) under ABI Prism 7900 sequence detection system (Applied Biosystems Life Technologies). The primers used in this study have been described previously [11 (link), 13 (link)]. The expression was quantified using the 2^−ΔΔCt method, and U6 and GADPH were used as internal controls.