Sepasol r rna 1 super g

The RNA of mesenteric fat, epididymal fat, and brown fat was extracted by Sepasol(R)-RNA I Super G (Nacalai Tesque, JAPAN) kit, measured at different absorbance values of 260, 280, and 320 nm, and then the concentration was calculated according to the absorbance value. Next, the RNA was transcribed into cDNA when configuring to 0.6 ug/ul. Specific primers were designed based on the sequence obtained from the literature and screened them through primer-BLAST website (Table 1), and then synthesized from Thermo Fisher Scientific (Waltham, MA, USA). Finally, THUNDERBIRD SYBR qPCR Mix was used to amplify the target gene (TOYOBO, Tokyo, Japan), and then all the prepared samples were kept into Thermal Cycler Dice (Takara Bio Inc. Japan). The cycle settings were as follows: 95°C, 30 s, 1 cycle; 95°C, 5 s, 60°C, 30 s, 40 cycles; 95°C, 15 s, 60°C, 30 s, 1 cycle, and the results were analyzed by 2^−ΔΔCt method.

Total RNA was isolated using Sepasol(R)‐RNA I Super G (Nacalai Tesque, Japan) from liver tissue, and the absorbance was measured at 260, 280, and 320 nm using a spectrophotometer. cDNA was synthesized with Rever Tra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Japan), in accordance with the manufacturer's protocol, and used for the amplification of target genes in real‐time PCR with the THUNDERBIRD SYBR qPCR Mix (TOYOBO, Japan). The specific primers were synthesized by Thermo Fisher Scientific (USA) (Table 2). The amplification was performed as follows in a Thermal Cycler Dice (TAKARA BIO INC., Japan): 1 cycle at 95°C for 30 s, and 40 cycles at 95°C for 5 s and 60°C for 30 s. We determined the fold differences in gene expression levels using the 2^−ΔΔCT method. The ratio of mRNA expression levels was normalized to the house keeping gene glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (Kudo et al., 2020 (link); Li et al., 2015 (link)). We show a comparison of gene expression by L‐Cit administration when the gene expression level in the control group is 100%. The notation of these data was taken from the published paper (Amengual et al.,l., 2018 (link)).