2.3. Gene Expression Analysis

RNA was isolated from epididymal white adipose tissue (F) using Qiagen RNeasy Lipid Tissue Mini Kit with on-column DNA digestion (Qiagen, Valencia, CA, USA). RNA (0.5 or 1 µg) was reverse transcribed into cDNA using an iScript cDNA Synthesis Kit from Bio-Rad (Bio-Rad, Hercules, CA, USA). PCR primers used in the real-time PCR analysis were previously published [28]. Analyses were performed on a Bio-Rad iCycler iQ thermocycler. Samples were analyzed in 30 µL reactions using SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA). All expression levels were normalized to the corresponding Rppl0 mRNA levels, and analyzed using the 2^−ΔΔCT method [43]. Rppl0 mRNA levels were unchanged in response to HFD or niacin treatment.