Quantitative Gene Expression Analysis

RNA was extracted from EAT and 3T3-L1 adipocytes using Sepasol (R)-RNA I Super G (Nacalai Tesque Inc., Kyoto, Japan). Absorbance of the extracted samples was measured at 260 and 280 nm using a branch photometer (Hitachi Ltd., Tokyo, Japan) to determine the RNA concentration. We performed reverse transcription to synthesize cDNA using Rever Tra Ace qPCR RT Master Mix and gDNA Remover (TOYOBO CO., LTD., Osaka, Japan). Then, real-time PCR was performed using THUNDERBIRD Next SYBR qPCR Mix (TOYOBO LTD., Osaka, Japan) and was used to determine the relative expression levels of each gene. For real-time PCR, we used specific primers synthesized by Thermo Fisher Scientific (Massachusetts, USA) (Table S2). RNA amplification was performed using a Thermal Cycler Dice (Takara Bio Co. Ltd., Shiga, Japan) based on the following protocol: 95°C for 30 s, 95°C for 5 s, and 60°C for 30 s. The relative expression levels of mRNA were determined using GAPDH as a reference housekeeping gene. The ratio of each transcript was calculated using the 2^−ΔΔCt method (63 (link), 64 (link)).