Computational protocol: High Salt Diet Induces IL 17 Dependent Gut Inflammation and Exacerbates Colitis in Mice

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Protocol publication

[…] Total RNA extraction from frozen colon of C57BL/6 mice that received either standard diet (control group) or HSD group for 3 weeks, 5 days, 3 days, 120 h, 48 h, 36 h, or 24 h and colon of mice that received a 50% NaCl solution by oral gavage and were euthanized after 90 min thereafter, was performed using TRIzol® reagent (Invitrogen, USA), followed by treatment with RQ1 RNase-Free DNase (Promega, USA), and cDNA synthesis using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, UK). qPCR assays were performed to evaluate the mRNA levels of Sgk1 (serum/glucocorticoid-regulated kinase 1) gene. Gapdh (glyceraldehyde-3-phosphate dehydrogenase) gene was used for the normalization of the data. Primers were designed using Primer3 program, version 0.4.0 (http://frodo.wi.mit.edu/) and analyses of parameters such as GC content and formation of homo-dimer, hetero-dimer, and hairpins structures were performed using OligoAnalyzer 3.1 software (https://www.idtdna.com/calc/analyzer). The specificity of the primers was confirmed using Primer-BLAST program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), to ensure that the primers amplified only the coding region (CDS) of interest. The sequences of the forward and reverse primers used for amplification of Sgk1 (GenBank: NM_001161847.2) and Gapdh (GenBank: GU214026.1) were 5′-CAAATCAACCTGGGTCCGTC-3′ (Sgk1-F), 5′-TCCAAAACTGCCCTTTCCG-3′ (Sgk1-R); 5′-CATCTTCCAGGAGCGAGACC-3′ (Gapdh-F), and 5′-GAAGGGGCGGAGATGATGAC-3′ (Gapdh-R). Each qPCR reaction contained 5 µL KAPA SYBR® FAST qPCR Master Mix 2× (Kapa Biosystems, USA); 0.2 µL of ROX High 50×; 200 ng of cDNA; each forward and reverse primer at the optimized concentrations (0.4 µM (F)/0.4 μM (R) for Sgk1 and 0.3 µM (F)/0.3 μM (R) for Gapdh) and water up to a final volume of 10 µL. The reaction profile was an initial step of denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 3 s and combined annealing and extension at 60°C for 30 s. A “no template control” was made in all the qPCR reactions for each pair of primers containing all the reagents except cDNA. Reaction specificity was confirmed with melting curves analysis and agarose gel electrophoresis experiments (Sgk1 amplicon size = 87 bp; Gapdh amplicon size = 150 bp). Standard curves were generated with series of log dilutions of cDNA to calculate the amplification efficiency (Sgk1: Eff = 98.98%, R2 = 0.99; Gapdh: Eff = 96.28%, R2 = 0.99). The qPCR reactions were performed using ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems, UK), and the data were processed by SDS Software, version 2.4 (Applied Biosystems, UK). The calculation of gene expression was performed using the 2−ΔCt method, where ΔCt = Ct value of target gene − Ct value of reference gene. […]

Pipeline specifications

Software tools Primer3, OligoAnalyzer, Primer-BLAST
Application qPCR
Organisms Mus musculus
Diseases Autoimmune Diseases, Cardiovascular Diseases, Colitis
Chemicals Sodium, Sodium Chloride