Rna mini kit

Total RNA was extracted using the Tri-RNA reagent or RNA mini kit (Favorgen, Taiwan) according to the manufacturer's instructions, and 500 ng of total RNA was used for the first cDNA synthesis (Toyobo, Osaka, Japan). The synthesized cDNA was diluted 10-fold, and mRNA expression was quantified using qRT-PCR.

RT-PCR was conducted to determine the expression level of the conserved regions of cmeA in all phenotypically tetracycline-resistant isolates. Briefly, total RNA was extracted using a RNA Mini Kit (Favorgen, Taiwan) according to the manufacturer's instructions, and subjected to cDNA synthesis using a RevertAid™ First Stranded cDNA Synthesis Kit (Fermentas, Lithonia). The 16SrRNA gene was used as an endogenous control. The PCR reaction mixture contained 12.5 µL of SYBR Green Master Mix, 400 ng of template DNA, 0.25 µM of forward and reverse primers each, and 12 µL of nuclease-free water. RT-PCR cycling conditions were as follows: 10 min at 50 °C, 5 min at 60 °C, and 5 min at 95 °C and then 40 cycles of 10 s at 95 °C, 30 s at 58 °C, and 15 s at 72 °C [29 (link), 30 (link)].

Isolated BAT was immediately frozen in liquid nitrogen and kept at −80 °C for further processing. Total RNA was isolated from BAT ground in liquid nitrogen using the RNA Mini Kit (Favorgen, Ping-Tung, Taiwan) according to the manufacturer’s instructions [72 (link)]. First-strand cDNA was synthesized with 550 ng of isolated total RNA and random hexamer using reverse transcriptase (Toyobo, Osaka, Japan) and mRNA expression was analyzed by real-time quantitative RT-PCR using a Light cycler480 (Roche, Basel, Switzerland) with Thunderbird SYBR green qPCR mix (Toyobo). Primers used for the amplification of specific genes are listed in the Supplementary Table S1. The expression of mRNA was normalized to that of L32.