Primerscript reagent kit

Trizol Reagent (Vazyme, NanJing, China) was used to extract total RNA from liver tissue, which was then treated by deoxyribonuclease I to remove the contaminant DNA. RNA was quantified based on the absorption of light by a Nanodrop ND-2000c spectrophotometer (Thermo Scientific, Camden, UK) at 260 nm (A260) and 280 nm. From each sample, 1 μg of RNA was used to synthesize cDNA in a 20 μL reaction mixture using the Primer-ScriptTM reagent kit (TaKaRa, Dalian, China) according to the manufacturers’ instructions. The real-time quantitative polymerase chain reaction was carried out by using the SYBR Premix Ex Taq II kit (TaKaRa) in an ABI 7300 fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA). The 20 μL reaction system included 10 μL of SYBR Premix Ex Taq buffer, 0.4 μL each of forward and reverse primers and dye, 2 μL of cDNA template, and 6.8 μL of distilled water. The real-time PCR cycling conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. The relative mRNA expression was determined using β-actin as an internal reference gene. The significance and correlation of quantitative results were analyzed by using 2‒ΔΔct as per Livak and Schmittgen [19 (link)]. Primer sequences are shown in Table 2.

Total RNA was extracted from liver samples using the Trizol reagent (TaKaRa Biotechnology Co., Dalian, China), according to the manufacturer’s instructions. The concentration of RNA in the final preparations was measured with a Nanodrop ND-2000c spectrophotometer (Thermo Fisher Scientific, Camden, USA). Reverse transcription was immediately performed using the Primer-ScriptTM reagent Kit (Takara Biotechnology Co., Dalian, China).
The expression of genes in liver was measured using real-time quantitative PCR (RT-qPCR) with SYBR Premix Ex Taq II kit (Tli RNaseH Plus; Takara Biotechnology) and an ABI 7300 Fast Real-Time PCR detection system (Applied Biosystems). The 20 μL reaction system included 2 μL of cDNA template, 0.4 μL of ROX reference dye (50×), 10 μL of SYBR Premix Ex Taq (2×), 0.4 μL of each forward and reverse primer, and 6.8 μL of double-distilled H₂O. The RT-qPCR cycling conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. All of the samples were run in triplicate, and the mRNA expression level of genes were calculated using the 2^−ΔΔCt method and normalized to the value of the reference gene β-actin. The final result of each target gene expression was expressed as the percentage of the CONT group. Primer sequences are shown in Table 2.

Samples of ileal mucosa were scraped on ice, frozen in liquid nitrogen immediately and stored at -80°C. Total RNA was extracted with Trizol reagents according to manufacturer’s instruction (Invitrogen, USA). RNA was quantified by nano-drop 2000 (absorbance ratios of 260/280 nm and 260/230 nm between 1.90 and 2.05) and verified by agarose gel electrophoresis. Then cDNA was prepared with Primer-Script^TM reagent kit, provided by TakaRa Biotechnology Co. Ltd (Dalian, China).
The gene-specific primers of occludin, claudin-2 and ZO-1 were synthesized by Invitrogen Biotech Co. Ltd (Shanghai, China) and are listed in Table 2. GAPDH was chosen as a housekeeping gene. Reverse transcription polymerase chain reaction (RT-PCR) tests were conducted with ABI 7600 RT-PCR system with a SYBR Premix Ex Taq^TM kits (TakaRa Biotechnology Co. Ltd Dalian, China). The relative mRNA expression was analyzed with ABI software and calculated with 2^-ΔΔCt as described previously [39 (link)].