Adipose Tissue RNA Extraction and Quantitative Gene Expression

Total RNA was isolated from adipose tissue (100 mg) using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Assessment of purity and quantity was measured with both a spectrophotometer (NanoDrop 1000, Thermo Scientific, Wilmington, DE, USA) and with a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). A starting sample of 300 ng of RNA was reverse transcribed for each case with the iScript kit (Bio-Rad, Hercules, CA, USA). Using TaqMan technology (Applied Biosystems, Waltham, MA, USA), quantitative real-time PCR was performed in triplicate on a 7500 instrument (Applied Biosystems) with the respective controls and quality checks included. Pre-designed exon-exon spanning TaqMan primer assays from Applied Biosystems were used (ID: TNF-α: Hs01113624_g1; SOCS3: Hs02330328_s1) and amplified in singleplex with the housekeeping gene peptidylprolyl isomerase A (PPIA: Hs99999904_m1) [48 (link)]. Gene expression was normalized using the 2−ΔCt method, including correction for amplification efficiency calculated from the standard curves for each primer set [49 (link),50 (link)]. As in previous reports [16 (link),17 (link),18 (link)], gene expression of PPIA showed no group differences in both VAT and SAT.