Determination of cytokine concentrations in the colonic tissue

Total cellular RNA was isolated using the GeneJET RNA Purification Kit (Thermo Scientific) according to manufacturer's instructions. The concentration of the isolated RNA was measured using the Qubit RNA BR assay (Invitrogen) for the Qubit 2.0 fluorometer. First strand cDNA was generated with the RevertAid First Strand cDNA Synthesis Kit (Thermo scientific) with 200 ng of the RNA templates. CDNA was stored at −20°C for later use. Quantitative real time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed with Applied Biosystems 7500 Real-Time PCR system and TaqMan gene expression products (Applied Biosystems) for IL-1β (assay ID: Rn00580432_m1), and TNF-α (assay ID: Rn01525859_g1) and, as control, ACTB (Rn00667869_m1). All reactions were conducted in triplicate, and each included a nontemplate control. Duplicate CT values were analyzed with the comparative CT (ΔΔCT) method (Applied Biosystems).