Quantitative Analysis of Colonic Gene Expression

RT-qPCR was applied to quantify the relative levels of MUC2, AQP3, and AQP8 mRNAs. After the isolation of total RNAs from mid-colon tissues using RNA Bee solution (Tet-Test Inc., Friendswood, TX, USA), cDNA was synthesized using reverse transcriptase (Superscript II, Thermo Fisher Scientific Inc.). The relative levels of three genes were quantified by the method described in a previous study using 2 × Power SYBR Green (Toyobo Co., Osaka, Japan) [48 (link)]. The primer sequences for the above analyses were as follows: MUC2, sense primer 5′-GCTGC TCATT GAGAA GAACG ATGC-3′, antisense primer 5′-CTCTC CAGGT ACACC ATGTT ACCAG G-3′; AQP3, sense primer 5′-GGTGG TCCTG GTCAT TGGAA-3′, antisense primer 5′-AGTCA CGGGC AGGGT TGA-3′; AQP8, sense primer 5′-CTGAG GCCCT CCCAC ATCT-3′, antisense primer 5′-GGAAA GGAAC AAGGC CAACA-3′; β-actin, sense primer 5′-ACGGC CAGGT CATCA CTATT G-3′, antisense primer 5′-CAAGA AGGAA GGCTG GAAAA GA-3′. The thermal cycling conditions consisted of the holding stage (1 min at 95 °C), cycling stage (40 cycles of 15 s at 95 °C, 15 s at 57 °C, and 45 s at 72 °C), and melt curve stage (15 s at 95 °C and 60 s at 60 °C). Additional analyses, including the fluorescence intensity, the threshold values, the threshold cycle (Ct), and the housekeeping gene, were conducted as described in a previous study [49 (link)].