Abi reverse transcription kit

Immortalized stromal vascular fraction (SVF) cells were exposed to DMEM/F-12 GlutaMAX (Invitrogen) containing dexamethasone (5 μM, Sigma), insulin (0.5 μg mL⁻¹, Sigma), isobutylmethylxanthine (0.5 mM, Sigma), rosiglitazone (1 μM, Cayman), T3 (1 nM, Sigma), and 10% FBS for 2 days. After induction, cells were maintained in media containing insulin (0.5 μg mL⁻¹), T3 (1 nM), 10% FBS, and 5 μM of each compounds for 3 days. Total RNA was extracted from 3T3-L1 cells or immortalized SVF cells. The RNA was reverse-transcribed using the ABI reverse transcription kit (Applied Biosystems/Thermo Fisher Scientific, Waltham, MA). Quantitative polymerase chain reactions were performed with SYBR green fluorescent dye using an ABI9300 polymerase chain reaction machine. Relative mRNA expression was determined by the ΔΔ^−C_t method normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Primer sequences are as follows. aP2 primers: 5′-AAG GTG AAGAGC ATC ATA ACC CT-3′ (forward), 5′-TCA CGC CTT TCA TAA CAC ATT CC-3′ (reverse); GAPDH primers: 5′-ACA CAT TGG GGG TAG GAA CA-3′ (forward), 5′-ACC CAG AAG ACT GTG GAT GG-3′ (reverse).