Optimized RNA Extraction and qRT-PCR Analysis

To extract high quality cellular RNA, we packaged the epididymal fat samples with aluminum foil, kept them at −70 °C, and used Mini RNA Isolation IITM (ZYMO Research, CA, USA). The samples were crushed in 300 μL of ZR RNA buffer, and only the supernatant was collected, centrifuged, and washed twice. After adding RNase-free water, we centrifuged the samples at 100 rpm and kept them at −70 °C [17 (link)].
We conducted qRT-PCR on AT samples using a 7900 HT Fast Real-Time PCR System (Applied Biosystems^®, Waltham, MA, USA) to quantify the expression of inflammatory genes, including TNF-α, F4/80, CCL2, C-C motif ligand 4 (CCL4), C-C motif ligand 5 (CCL5), regulated on activation, normal T-cell expressed, and secreted (RANTES)], and CXCR4. In the process of cDNA synthesis using Advantage RT PCR Kit (Clontech, Palo Alto, CA, USA), we cultured 1 μg of RNA, oligo (dT), and RNase-free H₂O mixtures at 70 °C for 2 min, kept them at 42 °C for 60 min, and mixed them with MMLV reverse transcriptase, 5× reaction buffer, recombinant RNase inhibitor, and 10 nM dNTP. We finally conducted RT-PCR with dH₂O, 2× SYBR reaction buffer, and specific primers (Table S2). We set the cycle threshold (Ct) for relative quantitation based on GAPDH using SDS Software 2.4 (Applied Biosystems^®) and adjusted the fold-change assuming that the value of the NC group was 1.