Gene Expression Analysis of Intestinal and Pancreatic Markers

Total RNA was extracted from the cecum and pancreas for gene expression analyses using the RNeasy Kit (Qiagen, Hilden, Germany), including an additional step of on-column DNase digestion (RNase-Free DNase Set, Qiagen, Hilden, Germany). Afterwards, cDNA synthesis was carried out using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. QuantiTect Primer Assays for claudin 2 (Mm_Cldn2_2_SG, Qiagen), claudin 4 (Mm_Cldn4_1_SG), claudin 8 (Mm_Cldn8_1_SG), mucin 2 (Mm_Muc2_2_SG), zonula occludens 1 (Mm_Tjp_1_SG), aquaporin 1 (Mm_Aqp1_1_SG), and aquaporin 4 (Mm_Aqp4_1_SG) were used to perform qPCR analyses. Beta actin (Mm_Actb_2_SG) served as an endogenous control. Gene expression analyses for facilitated glucose transporter (Slc2a5, Mm_00600311_m1), sodium/glucose cotransporter (Slc5a1, Mm_00451203_m1), and trypsinogen (Prss2, Mm_00657001_m1) were performed using TaqMan^® Gene Expression Assays and beta actin (Mm_00607939_s1) as a reference gene. Detection was performed with the QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Weiterstadt, Germany) using the Fast SYBR Green^® Master Mix or TaqMan^® Fast Advanced Master Mix according to the manufacturer’s instructions. All reactions were run in triplicates. The amplified PCR product was verified by a melting curve analysis (for SYBR green chemistry). Samples were additionally normalized to the reference sample generated from mixed cDNA isolated from proximal colon, ileum, mesenteric lymph nodes, liver and spleen. Relative gene expression was calculated using the 2^−ΔΔCt method using the Thermo Fisher Connect^TM platform (Thermo Fisher Scientific, Waltham, MA USA).