Computational protocol: G Protein coupled pH sensing Receptor OGR1 Is a Regulator of Intestinal Inflammation

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Protocol publication

[…] Global whole-transcript analysis was performed using a GeneAtlas microarray system (Affymetrix) to compare response differences between Ogr1+/+ and Ogr1−/− murine macrophages after 24 hours acidic pH shift. Mature murine quiescent peritoneal macrophages were isolated as described above, from age-matched female Ogr1−/− and Ogr1+/+ mice (C57BL/6). Five replicates or mice per condition were used, and approximately 1 × 106 macrophages per mouse obtained. Cells were not pooled. Cells were treated with pH 6.7 equilibrated medium to activate Ogr1, and pH 7.7 to serve as negative controls. Cells were collected, and RNA and cDNA samples were prepared. Biotin-labeled cDNA samples were hybridized to GeneChip Mouse Gene 1.1ST Array Strip (Affymetrix, P/N 901628) after protocols provided by Affymetrix. Data were summarized on gene-level using RMA (Robust Multiarray Average). Data quality was assessed using the bioconductor/R package “arrayQualityMetrics,” and reproducibility was assessed using Pearson's correlation for all the filtered expression values and hierarchical clustering. For all pairwise comparisons, differentially expressed genes were selected using ≥2.0-fold change, P < 0.05 significance, as determined using analysis of variance (as implemented by the R package, Linear Models for Microarray Data, “limma”) and F-test for the complete experimental design. The results were analyzed by global ranked fold change and using Metacore software for pathway enrichment. […]

Pipeline specifications

Software tools arrayQualityMetrics, limma, MetaCore
Databases GeneAtlas
Application Gene expression microarray analysis
Organisms Mus musculus, Homo sapiens
Diseases Deficiency Diseases