Power sybr green 2 mastermix

White adipose tissue (WAT) was collected at the week 18 necropsy from the intra-abdominal perigonadal depots. Tissue from the four heaviest animals in each treatment group was selected to capture the effects in animals most sensitive to weight gain. A range in sensitivity was expected due to the genetic variation in the outbred CD-1 mice used in the study. RNA was isolated by homogenizing WAT with an automated ceramic bead homogenizer FastPrep-24 5G (MP Biomedicals, Irvine, CA) in Trizol (Thermofisher Scientific, PA, USA) for 2 cycles of 4 m/s for 30sec. RNA was purified using a Qiagen RNeasy kit (Qiagen, MD, USA) and genomic DNA was removed using on-the-column DNase I digestion (Qiagen, MD, USA) and quantified with Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, PA, USA). RNA (1 mg) was transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). cDNA (25 ng) was amplified in triplicate using a QuantStudio 7 Flex Real Time PCR System (Applied Biosystems) and Power SYBR Green 2 × Mastermix (Applied Biosystems, Foster City, Canada). Primer sets for all genes were pre-designed assays (IDT DNA, Redwood City, CA, USA) and are shown in Table S2. Mean C_t values were normalized to Rpl19 (60S ribosomal protein L19; housekeeping gene) and relative fold change mRNA levels were calculated by using ΔΔ C_T method [43 ].