Nanodrop microvolume uv vis spectrophotometer

Total RNA was isolated using RNA Pure kits (TR-205-50, ZYMO RESEARCH) according to the manufacturer’s guidelines from either cultured cells or liquid nitrogen–frozen tissues. The concentration and purity of each RNA extract were checked at a 260/280 ratio on a NanoDrop Microvolume UV-Vis Spectrophotometer instrument (Thermo Fisher Scientific, One^c). Complementary DNA (cDNA) synthesis was achieved by using the PrimeScript^TM RT Reagent Kit (RR037A, TaKaRa) according to the manufacturer’s guidelines. Quantitative PCR was performed with SYBR™ Select Master Mix (4472908, Applied Biosystems) following the product’s instructions. Primer sequences were listed in Appendix Table S4. Three technical replicates were set for every single reaction to ensure reliability and validity. The ΔΔCT value of the target gene expression was measured and assessed against the value of the reference genes GAPDH and U6.

The Kupffer cells underwent lysis through the utilization of pre-cooled Trizol on ice for a duration of 5 min, followed by the extraction of their total RNA via chloroform. The RNAs were then precipitated with isopropanol and resuspended in 75% pre-cooled ethanol. The RNA concentration was assessed by means of NanoDrop Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) after dissolution in diethyl pyrocarbonate (DEPC)-treated water. Reverse transcription was performed using the ReverTra ACE qRT-PCR RT Kit (TOYOBO, FSQ-101, Tokyo, Japan), and the resulting cDNA was quantified using Power Green qRT-PCR Mix (Dongsheng Biotech, P2102, Guangzhou, China) under 2^−ΔΔCt method. The primer sequences are given in Table S1. Ultimately, 18S, β-actin and U6 were wholly selected as the internal controls.

Using RNA Pure kits (Zymo Research, TR‐205‐50), total RNA was extracted in accordance with the manufacturer's instructions. The concentration and purity of the extracted RNA were assessed using a NanoDrop Microvolume UV/Vis Spectrophotometer (Thermo Fisher Scientific). To produce the necessary complementary DNA, the extracted RNA was then reverse transcribed using the PrimeScriptTM RT Reagent Kit (TaKaRa, RR037A) in accordance with the kit's instructions. SYBR™ Select Master Mix (Applied Biosystems, 4472908) was used to create a 20 μL total volume of the real‐time (RT)‒PCR system in accordance with the manufacturer's recommendations. RT‒PCR was carried out using a QuantStudio 3 Real‐Time Fluorescence PCR System (Applied Biosystems) on the samples to be analyzed. The relative expression of mRNA in the cells was determined using the 2−ΔΔCt method. The data of three biological duplicate experiments are shown in these histograms. All the data are presented as the mean ± SD of three experiments. In Table S2, a list of primer sequences is provided. For each reaction, three technical duplicates were performed to guarantee reliability and validity. The CT value of the target gene was calculated and compared to that of the reference gene glyceraldehyde 3‐phosphate dehydrogenase (GAPDH).