Thermo scientific nanodrop lite

Gene expression analysis was conducted by employing quantitative real-time polymerase chain reaction (q-RT-PCR).
Total RNA isolated from the aorta and jejunum underwent quantification using Thermo Scientific™ NanoDrop Lite (Thermo Fisher Scientific). Subsequently, all samples were diluted to a concentration of 5 ng/μL with DNase–RNase-free water. The relative expression levels of each target gene in the aorta (Mcp1(Ccl2), Il1b, Ifng, Tnfa, Il33, Il4, Il5, Icam1, and Vcam1), jejunum (Cd36, Il6, Pept1, Sglt1, and Tnfa), and epididymal white adipose tissue (eWAT) (Mcp1(Ccl2), Il1b, Ifng, and Tnfa) were normalized by utilizing the Gapdh threshold cycle (CT) values and quantified by employing the comparative threshold cycle 2^−ΔΔCT method, as previously outlined [24 (link)].
Given the predominant site of fatty acid absorption in the small intestine, we examined Cd36 and Il22 gene expression levels in the jejunum instead of the large intestine, aiming to explore alterations in fatty acid absorption. Nine mice from each group were assessed, with RT-PCR conducted in triplicate for each sample.

Total RNA was extracted using a TRIzol reagent according to the manufacturer’s instructions. Thermo Scientific ™ NanoDrop Lite (Thermo, Waltham, MA, USA) was used to measure the integrity and concentration of RNA in the samples, and the subsequent experiments were performed after passing. Total RNA was stored at −80 ° C. Prime Script RT Master Mix Perfect Real-Time (Takara, Dalian, China) was used for mRNA cDNA synthesis and the miRNA reverse transcription reaction was performed using a one-step miRNA cDNA synthesis kit (HaiGene, Haerbin, Heilongjiang China).
The real-time PCR primer sequences were shown in Table S7 and designed by the Primer Premier 6 software (PREMIER Biosoft, San Francisco, CA, USA). The CFX96-Touch^TM real-time PCR detection system (Bio-Rad, Hercules, CA, USA) was used to detect the mRNA abundance. The reaction volume of real-time PCR includes 1 μL cDNA, 0.5 μL reverse and forward primers (per gene), 3 μL double distilled water, and 5 μLTB Green™ Premix Ex Taq™ II (Takara). All reactions were repeated three times. Relative gene expression was determined by the 2^−ΔΔCt method [44 (link)].