Optimized Quantification of Gene Expression in Colonic Tissue

RNA from the proximal colonic section was isolated using a QIAGEN RNeasy Blood and Tissue kit according to the manufacturer’s instructions. The integrity and purity of RNA were verified by agarose gel electrophoresis and spectrophotometry using a NanoDrop ND-1000. RNA was reverse transcribed by SuperScript III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative PCR (qPCR) was carried out with EvaGreen intercalating dye (Syntol, Moscow, Russia) on the CFX96 Touch™ Real-Time PCR Detection System (BioRad, Hercules, CA, USA). PCR conditions included a preheating step at 95 °C for 3 min and 40 cycles at 95 °C for 15 s, 57 °C for 15 s, and 72 °C for 15 s coupled with fluorescence measurement. The amplification specificity was confirmed by melt curves analysis. Each sample was run in triplicate, and a non-template control was added to each run. The choice of reference genes TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) was based on the results of a stability study of the reference genes in a mouse model of DSS-induced colitis [33 (link)]. The expression levels of the target genes were determined by the ΔΔCt method with the calculated primer efficiencies and normalized on the geometric mean of selected reference genes. Primer sequences are listed in Supplementary Table S2.