Quantitative Real-Time PCR

Total RNA was extracted from the subcutaneous white adipose tissue with TRIzol^® reagent (Invitrogen, California, USA). Reverse transcription was conducted with a SuperScript First-Strand cDNA Synthesis kit (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT–PCR) was performed with SYBR Green Master Mix (Roche Applied Science, Mannheim, Germany). Gene expression was detected with a 7500 real-time PCR system (Applied Bioscience, Foster City, CA, USA), and the thermal settings used were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 65°C for 1 min. The primers (Shanghai Life Biotechnology Co., Ltd., Guangzhou, China) used are listed as following: TNF-α, forward primer: 5’- TGCCTATGTCTCAGCCTCTTC-3’, reverse primer: 5’- GGTCTGGGCCATAGAACTGA-3’; Cd68, forward primer: 5’-TGTCTGATCTTGCTAGGACCG-3’, reverse primer: 5’-GAGAGTAACGGCCTTTTTGTGA-3’; Adgre1, forward primer: 5’-TGACTCACCTTGTGGTCCTAA-3’, reverse primer: 5’-CTTCCCAGAATCCAGTCTTTCC-3’; Adipoq, forward primer: 5’-TGTTCCTCTTAATCCTGCCCA-3’, reverse primer: 5’-CCAACCTGCACAAGTTCCCTT-3’. Gapdh was utilized for normalization. Data were analyzed using the 2^-ΔΔCt relative expression method.