Thunderbird sybr quantitative pcr mix

The hypothalamus, pituitary, iWAT, BAT, and liver were dissected from the mice, snap-frozen in liquid nitrogen, and stored at −80°C for RNA processing at the endpoint of the infusion of RFRP-3. The total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) for brain and liver tissues or QIAzol lysis reagent for iWAT and BAT (QIAGEN, Venlo, Netherlands) following the manufacturer’s instructions. The RNA concentration was measured by nanodrop spectroscopy (Thermo Fisher Scientific, Waltham, MA, USA), and the first-strand cDNA was synthesized from the total RNA using a ReverTra Ace kit (TOYOBO, Osaka, Japan). The primer sequences used in this study are listed in Table 2. The PCR amplifications were performed with THUNDERBIRD SYBR quantitative PCR mix (TOYOBO) using the following conditions: 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s, and 60 °C for 30 s. The PCR products in each cycle were monitored using a Bio-Rad CFX Connect (Bio-Rad Laboratories, Hercules, CA, USA). The relative quantification of each gene was determined by the 2^–∆∆Ct method using β-actin (Actb) for brain and liver tissues or ribosomal protein S18 (Rps18) for iWAT and BAT as internal controls [60 (link)].

Quantitative real-time PCR was performed to determine mRNA expression levels. Total RNA was extracted from muscle using Isogen II (Nippon Gene, Tokyo, Japan), according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA using a ReverTra Ace kit with genomic DNA remover (Toyobo, Tokyo, Japan). Real-time PCR was performed with Thunderbird SYBR quantitative PCR mix (Toyobo, Tokyo, Japan) and CFX96 Touch real-time PCR detection system (Bio-Rad, Tokyo, Japan). The expression levels of selected genes were analyzed using standard curve method and the values were normalized against TATA box binding protein (TBP). Primer sequences were as follows: TBP [forward(F) 5′-CAGATGTGCGTCAGGCGTTC-3′ and reverse (R) 5′-TAGTGATGCTGGGCACTGCG-3′]; Zmynd17 (F 5′-TAGGGCTTAACAGGCACTGGTCCCC-3′ and R 5′-TTCTTGTGCTTTCGCCGCCGTG-3′).