Mcp1 mm00441242 m1

Total RNA was extracted with the RNAeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany). RNA concentration was determined spectrophotometrically using NanoDrop® (Thermofisher). RNA (0.25–1 µg) was reverse transcribed with PrimeScript RT reagent kit using random hexamer primers (Takara, Kusatsu, Japan). TaqMan system was used for real-time PCR amplification. Relative gene expression was obtained after normalization to 18s RNA, using the formula 2^−ΔΔcp. The following primers/probes were used: Tnfα Mm00443258_m1, Il-6 Mm00446190_m1, Il-1β Mm0043422/8_m1, Mcp1 Mm00441242_m1, Il-10 Mm00439614_m1, F4/80 Mm00802529_m1, Ask1 Mm0043883_m1, 18 s 4352930, Ucp1 Mm01244861_m1, Pgc1α Mm01208835_m1, Cidea Mm00432554_m1, Prdm16 Mm00712556_m1 (Applied Biosystems, Rotkreuz, Switzerland).

Total RNA was extracted for reverse transcription, and real-time quantitative RT-PCR analysis was performed on an ABI PRISM 7900 sequence detector (Applied Biosystems, Foster City, CA, USA) as previously described [23] (link). Primers and probes for interleukin (IL)-1β (TaqMan Gene Expression Assay ID Mm00434228_ml), IL-6 (Mm00446190_ml), monocyte chemoattractant protein-1(MCP-1, Mm00441242_m1), and macrophage inflammatory protein-2 (MIP-2, Mm00436450_m1) were purchased from Applied Biosystems. β-Actin (Rn00607939_s1) was used as endogenous control. Thermal cycling was initiated with a 2-min incubation at 50°C, followed by a 10-min denaturation step at 95°C and 40 cycles at 95°C for 15 s and 60°C for 1 min. Relative quantities of the candidate genes and β-actin mRNA were calculated using the comparative threshold cycle (Ct) method.

Total RNA was isolated from frozen IWAT and EWAT samples, as previously described. Briefly, frozen tissue was homogenized in Trizol reagent and phase separation was carried out using chlorofom per the manufacturer’s protocol (Sigma Aldrich, St. Louis, MO, USA). 70% ethnaol was added to the aqueous layer, which was then further purified using a RNeasy mini kit (Qiagen, Valencia, CA, USA) per the manufacturer’s protocol. RNA quantity and quality were analyzed by spectrophotometry (NanoDrop, ND-1000, Thermo Scientific, Wilmington, DE, USA), and a cDNA library was created using M-MLV reverse transcriptase (Promega, Madison, WI, USA) per the manufacturer’s protocol.
qRT-PCR was performed using commercially available Applied Biosystems Taqman primers and probes (Cd11b, Mm01271255_m1; Cd68, Mm04411920_m1; F4/80, Mm00802527_m1; Mcp-1, Mm00441242_m1; Applied Biosystems, Foster City, CA, USA) and samples were run in duplicate on the ABI 7900HT platform (Applied Biosystems, Foster City, CA, USA) using ABI Taqman Universal MasterMix (Applied Biosystems, Foster City, CA, USA). Gene expression was analyzed using a standard curve and normalization to cyclophilin as the endogenous control. Data are expressed as arbitrary units (AU).