Rt pcr instrument

RAW 264.7 cells were seeded in 6-well plates at a consistency of 1 × 105/well and cultured with DHT (5 μM and 10 μM) and RANKL (50 ng/ml) for 6–7 days, with medium change performed every 2 days until the formation of mature osteoclasts. Total RNA was extracted by TRIZOL (Thermo Fisher Scientific, China) and the obtained RNA was reverse transcribed into cDNA by EVOM-MLV Premix Kit. PCR was performed using SYBR Green Kit and the following cycling conditions: 95°C, 30 min; 95°C, 5 s; 60°C, 30 min; and 50 cycles. All primers are listed in Table 1. All relevant gene expression results were processed using the RT-PCR instrument (Bio-Rad, United States). The 2^−ΔΔCT method was used to calculate the comparative expression levels of each gene.

Total RNA from BAT of 5 h fasted mice was isolated using a RNeasy lipid tissue mini kit (Qiagen, Hidden, Germany) according to the manufacture’s protocol, and first-strand complementary DNA (cDNA) was synthesized from 1 µg total RNA using a iScript cDNA synthesis kit (Bio-Rad Laboratories) [60 (link)]. The sequences of the primers (Integrated DNA Technologies, Coralville, IA, USA) were according to our published protocols [32 (link),42 (link)]. Quantitative PCR (qPCR) was performed in a 25 µL final reaction volume, including 4µL of 10-fold diluted sample cDNA, 1µL of 10µM forwarded or reverse primers, and iTaq universal SYBR green supermix (Bio-Rad Laboratories), using a Bio-Rad RT-PCR instrument. PCR conditions were conducted as follows: 95 °C for 10 min for one cycle, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Threshold cycle readings for each of the unknown samples were obtained, and the results were analyzed in Excel using the ∆∆Ct method [35 (link)]. Levels of 36B4 mRNA from each sample were similar among all groups and were used as internal controls to normalize the mRNA levels.