Column rna extraction kits

The iWAT and BAT were homogenized and total mRNA was extracted using column RNA extraction kits (Magen, Guangzhou, China, R4121). The total RNA concentration was determined using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the mRNA was reverse transcribed to cDNA using cDNA synthesis kits (CWBIO, Jiangsu, China, CW2582M). Information on the primers is shown in Supporting Information (Table S6) and synthesized (Sangon Biotech, Shanghai, China). qPCR was performed in an Applied Biosystems by Life Technologies QuantStudio 7 (Thermo Fisher Scientific). The relative abundance of the genes was normalized to GAPDH using the 2-ΔΔCT method and the relative expression level were shown as fold changes relative to chow group.

Total RNA extraction, cDNA synthesis, and qPCR were performed using previously described methods (5 (link)). Briefly, total RNA was extracted from 0.1 g of liver or ileal tissue using column RNA extraction kits (Magen, Guangzhou, China). RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 1% agarose gel electrophoresis, respectively. Approximately 1 μg of the isolated RNA was reverse transcribed into cDNA using a cDNA synthesis kit (CWBIO, Jiangsu, China) following the manufacturer’s instructions. qPCR was conducted on an ABI 7900HT Real-Time PCR System (Applied Biosystems, Branchburg, NJ, USA). The primers used in the study were designed with Primer3 and BLAST using default parameters (Premier Biosoft International, Palo Alto, CA, USA) and are described in Supplementary Table S2. The PCR cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s, and then 1 cycle of 72°C for 30 s. Relative gene expression levels were calculated using the 2^–ΔΔCt method after normalization to GAPDH or β-actin (18 (link)).