RNA Isolation and Real-Time Quantitative PCR

Total RNA was isolated from epididymal adipose tissue by using the RNeasy Lipid Tissue Mini Kit following the manufacturer’s protocol (Qiagen, Germantown, MD, USA). The quality and quantity of RNA were analyzed by using Nanodrop 8000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). cDNA was synthesized by using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) following the manufacturer’s protocol. Real-time qPCR of Mcp-1 was analyzed and normalized to the 18s rRNA by using the TaqMan Assay of Demand primers on the ABI QuantStudio 12K-Flex Real-time PCR system (Applied Biosystems). The 2^-ΔΔCT method was used to calculate the relative changes in gene expression (31).