Intestinal cytokine mRNA detection

Intestinal expression of cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-23, IL-10, and IL-1β was evaluated. Total RNA was isolated from 100 mg of ileal and colonic tissues by using a RNA Extraction Kit (Takara, Japan) and processed with a PrimeScript RT reagent Kit (Takara, Japan) to synthesize cDNA. Primers used are listed in Table ​Table1.1. Quantitative real-time PCR was performed in triplicate for each sample with Lightcycler 480 instrument (Roche Applied Science, Penzberg, Germany). Reaction conditions for amplification of DNA were as follows: 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Cytokine transcript levels were normalized with β-actin, and relative gene expression was expressed as the fold change (2^-ΔΔCt) relative to the expression in the control samples.

Primer sequences

IL: Interleukin; TNF: Tumor necrosis factor.