Trizol reagent kit

Total RNA extraction was done using the Trizol reagent kit (Accurate Biotechnology Co., Ltd., Hunan, China), the 1-step gDNA removal kit (TransGen Biotech Co., Ltd., Beijing, China) was used to synthesize cDNA, and the RT-PCR was performed as our previously described method (Lan et al., 2014 (link)). The primer sequences used were listed in Supplemental Table S3. The 2^−ΔΔCt method was used to calculate the relative gene expression, and β-actin used as internal reference gene (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted from the liver, jejunum mucosa and ileum mucosa using the TRIZOL reagent kit (Accurate Biology, Changsha, China). Then cDNA synthesis from 500 ng RNA was performed based on Evo M-MLV RT Premix for qPCR (Accurate Biology). Gene expression of inflammatory cytokines and tight junction protein were quantified by RT-PCR. The assay was carried out using SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology) on a Roche-LightCycler 96 instrument (Basel, Switzerland). Genes expression was standardized by β-actin, and the method of 2^–△△Ct was used to calculate the relative expression of each gene. Primer sequences used in the current study are listed in Table S3.