Qiazol rneasy lipid tissue mini kit

Total RNA was extracted from ATs with the Qiazol RNeasy Lipid Tissue mini kit (QIAGEN, Valencia, CA, USA) or with RNA-Stat 60 Reagent (Ams Biotechnology, Abingdon, UK) for 3T3-L1 cells, according to the manufacturer’s recommendations. Then, 1, 2, or 4 μg of total RNA was converted to cDNA using RT (Transcriptor Reverse Transcriptase 20 U/μL, 03531287001; Roche). Next, qRT-PCR amplification was performed using specific TaqMan Gene Expression Assays, 10 ng of cDNA, and Brilliant III Ultra-Fast qPCR Master Mix. Specific signals were normalized to the constitutively expressed TATA sequence binding protein (TBP; Mm 00446973; Thermo Fisher Scientific) for AT using the formula 2^−Δct or to β-actin (mouse ACTB, 452341E; Thermo Fisher Scientific) for 3T3-L1 cells using the formula 2^−ΔΔct. The TaqMan probes used in this study are Vegf-A (Mm 00437306-m1), Vegf-B (Mm 00442102_m1), Ucp1 (Mm 01244861-m1), Tmem26 (Mm 01173641-m1), Pgc-1α (Mm 01208835), Prdm16 (Mm 00712556-m1), Cidea (Mm 00432554-m1), Ppar-γ (Mm 00440940-m1), P53 (Mm01731290-g1), Tgf-β1 (Mm01178820-m1), Fgf21 (Mm 00840165-g1), Hoxc9 (Mm 00433972-m1), Sirt1 (Mm01168521-m1), Mmp-9 (Mm 00442991_m1), Timp-2 (Mm 00441825_m1), Timp-3 (Mm 00441826-m1), Cebp-α (Mm 00514283 s1), Bcl-2 (Mm 00477631-m1), Bid (Mm 00432073_m1), and Casp-3 (Mm 01195085-m1).