RNA analysis

Approximately 1-cm (in length) samples from distal parts of the colon were disrupted and homogenized with the TissueLyser LT (Qiagen, Hilden, Germany) followed by total RNA extraction according to the manufacturer's instruction using RNeasy Plus Universal Mini Kit (Qiagen, Hilden, Germany) and chloroform (Sigma-Aldrich, St. Louis, MO). No further treatment with DNase was needed because gDNA Eliminator Solution is included in the kit.

For cDNA synthesis, 5 μg of total RNA was used. Reverse transcription was performed in a Mastercycler gradient (Eppendorf, Hamburg, Germany) using QuantiNova Reverse Transcription kit (Qiagen, Hilden, Germany). Samples were diluted with RNase-free water to obtain a cDNA concentration between 10 pg and 100 ng as required by the TaqMan Fast Advanced Master Mix protocol (Thermo Fisher Scientific, Waltham, MA).

RNA and cDNA purity was assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

According to the TaqMan Fast Advanced Master Mix protocol (Thermo Fisher Scientific, Waltham, MA) quantitative RT-PCR was performed using the Applied Biosystems StepOnePlus RT-PCR system (Thermo Fisher Scientific, Waltham, MA). Single-tube TaqMan gene expression assays (Thermo Fisher Scientific, Waltham, MA) included the housekeeping genes GAPDH (Mm99999915_g1) and GUSB (Mm00446953_m1), as well as TGFβ (Mm 01178820_m1), HGF (Hs04329698_m1), CCL17 (Mm01244826_g1), IFNγ (HS00989291_m1) and TNFα (HS01113624_g1) (Thermo Fisher Scientific assay IDs are given in brackets). Analysis was performed using StepOnePlus™ Software v2.3. For expression analysis, a mean value of cycle threshold values was calculated for two housekeeping genes. Relative expression values for the respective analyzed genes were calculated as the difference between the mean cycle threshold (CT) value of the housekeeping genes and the respective analyzed gene (delta CT). Relative expression is depicted as the logarithmic value of the delta CT.