Quantifying Gene Expression in Adipose Tissue

Total RNA of adipose tissue was obtained using RNeasy lipid tissue (Qiagen, Hilden, Germany). cDna was synthesized using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). PCRs were performed with a Bio-Rad CFX96 Connect real-time PCR system instrument and software (Bio-Rad Laboratories srl, Milan, Italy), as previously reported [21 (link)]. Each cDNA sample (500 ng) was mixed with 2× QuantiTech SYBRGreen PCR Master Mix (Qiagen, Hilden, Germany,) and validated primers (Appendix A, Table A1). Data were normalized to Actb for BAT and Rn18S for scWAT as a housekeeping gene, and the data were analyzed according to 2^−ΔΔCt method.