Selected mRNA abundance was determined by RT-qPCR, including nutrient transporters genes FABP-1 (fatty acid binding protein 1), FATP-1 (fatty acid transport protein 1), GLUT-2 (glucose transporter 2), LAT-1 (L type amino acid transporter 1), PepT-1 (peptide transporter 1) and SGLT-1 (sodium glucose co-transporter 1), inflammatory molecules’ genes TLR-4 (Toll-like receptor 4), IL-1β (interleukin 1β), IL-8, IL-10, TNF-α (tumor necrosis factor α), TGF-β (Transforming growth factor β) and NF-κB (Nuclear factor kappa B), and tight junction genes Claudin-1, Occludin, ZO-1 and Mucin-2. Total RNA was isolated from ileal mucosa samples (approximately 0.75 mg) using an RNAprep pure tissue kit (Tiangen Biotech Co. Ltd., Beijing, China) under the manufacturer’s instructions. Total RNA concentrations and quality were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was evaluated using agarose gel (1%) electrophoresis. Then, cDNA was synthesized from 1 μg total RNA using a PrimeScript RT reagent kit (TaKaRa Biotechnology Co., Ltd., Otsu, Japan) following to manufacturer’s protocols. Selected mRNA reactions were detected in 10 μL (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR® Premix Ex TaqTM II (Tli RNaseH Plus) (TaKaRa Biotechnology Co., Otsu, Japan). The primers for nutrient transporters, inflammatory, and tight junction-related and housekeeping genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] were described previously (Wang et al., 2016 (link), 2020 (link)). The 2–ΔΔCt method was used for quantification using GAPDH as a reference gene, and relative abundance was normalized to CON group values.
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