Computational protocol: Inhibition of miR-142-5P ameliorates disease in mouse models of experimental colitis

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Protocol publication

[…] Total RNA was isolated using Trizol, according to manufacturer’s protocol (Trizol reagent, Invitrogen, USA). Quantity and quality of the RNA was checked by the Agilent 2100 Bioanalyzer platform (Agilent Technologies, Inc., Santa Clara, CA, U.S.A.). RNA integrity numbers (RIN) values of our samples ranged between 8.7 and 9.6. RNA concentration was measured using the Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, U.S.A).For the miRNA microarray experiment, samples were hybridized overnight onto miRXplore™ microarrays (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) at Miltenyi. Experimental and control samples (miRXplore universal reference, UR) were labeled with Hy5 and Hy3 respectively. Signal intensities were obtained using the ImaGene software (Biodiscovery) and normalized mean Hy5/Hy3 ratios were calculated using the PIQOR analyzer. The normalized Hy5/Hy3 ratios were log2-transformed. A systematic shift towards positive log-ratios on all arrays was corrected by fitting an equal variance two-component Gaussian mixture model (R package Mclust[]) per array and subtracting the mean corresponding to the positive mode of the mixture model.For the mRNA microarray experiment in experiment #1, additional quality control, RNA labeling, hybridization and data extraction were performed at Miltenyi Biotec GmbH (Bergisch Gladach, Germany). A total of 825ng of biotinylated cRNA was hybridized overnight onto the Agilent Whole Mouse Genome Oligo Microarray 4x44, using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Microarrays were scanned using an Agilent dual-laser DNA microarray scanner. The default Agilent normalization procedure, called Linear & Lowess, was applied. Normalized data were imported in Rosetta Resolver (Rosetta Biosoftware). An error-weighted 1-way ANOVA with Benjamini-Hochberg false-discovery rate multiple testing correction was used to identify differentially expressed genes between transfer-colitic and control mice at each of the three time points.Amplification, labeling, hybridization and data extraction of material of experiment #4 were performed at ServiceXS (Leiden, The Netherlands) following the manufacturer's protocol. 1500ng of biotinylated cRNA was hybridized onto the Illumina MouseWG-6 v2 BeadChip (Illumina, Inc., San Diego, CA, U.S.A.). Hybridization and washing were performed according to the Illumina Manual “Direct Hybridization Assay Guide”. Scanning was performed on the Illumina iScan (Illumina, Inc., San Diego, CA, U.S.A.), while data extraction was performed with Illumina GenomeStudio v2011.1. Normexp-by-control background correction, quantile normalization, and log2 transformation[] were performed on the Illumina sample and control probe profiles using the limma R package (version 3.10.2). The arrayQualityMetrics R package (version 3.10.0) was used to assess whether the microarray data were of good quality. Only probes detected (detection p-value < 0.05) on at least one array were included in the differential expression analysis. The illuminaMousev2.db R package (version 1.12.2) was used to update the probe annotation provided by Illumina.For both the miRNA (#1) and the mRNA microarray experiment (#4), gene-wise linear models were fitted using the limma R package. Differential expression between transfer-colitic and control mice at each of the three time points and between anti-miR-142-5p and scrambled anti-miR treated mice, respectively, was assessed via an empirical Bayes moderated t-test. Ingenuity Pathway Analysis (IPA Spring Release 2016, QIAGEN) was used to identify upstream transcriptional regulators. […]

Pipeline specifications

Software tools ImaGene, mclust, GenomeStudio, limma, arrayQualityMetrics, IPA
Application Gene expression microarray analysis
Organisms Mus musculus, Homo sapiens
Diseases Colitis, Inflammatory Bowel Diseases, Severe Combined Immunodeficiency, Wasting Syndrome