2. Extraction of total RNA from the proximal epididymal fat and generation of complementary DNA (cDNA)

One milliliter of TRI REAGENT solution (Molecular Research Center, Inc, Cincinnati, OH) was applied to each proximal epididymal fat sample for homogenization. Then, chloroform and isopropanol were sequentially added to extract total RNA from tissue homogenate, and an acquired total RNA pellet was re-dissolved in DEPC-dH₂O. The yield and concentration and quality of total RNA were evaluated by the NanoDrop Lite spectrophotometry (Thermo Scientific, Wilmington, DE) and 1.2% agarose gel electrophoresis, respectively.

One microgram of total RNA was used to construct the first-stranded complementary DNA (cDNA) by reverse transcription (RT) reaction in iScript^TM Reverse transcription Supermix for RT-qPCR (Bio-Rad Laboratories. Inc., Hercules, CA). The total RNA was mixed with oligo-dT primer, RTase, 5X buffer, and nuclease-free dH₂O, and the final volume of a RT reaction was 20 μL. The RT reaction was performed in 25°C for 5 min, 46°C for 20 min, and 95°C for 1 min, and generated cDNA was directly utilized for quantitative real-time polymerase chain reaction (PCR).