qPCR-based Gene Expression Analysis

Cells were collected in Trizol reagent (Thermo Fisher Scientific), and RNA was isolated manually by chloroform extraction and isopropanol precipitation. RNA concentrations and purity were determined by spectrophotometry. cDNA was generated by a TaqMan™ reverse transcription reagents kit (Thermo Fisher Scientific). The gene primers and probes were designed by Applied Biosystems. Gene expression was determined by qPCR (∆Ct method), as per the described protocols [29 (link),30 (link)]. All samples were run in triplicates. Human GAPDH was used as an endogenous control. Quantitative PCR experiments were repeated at least five times with SVFs from independent healthy donors, or with SGBS samples from independent passages.