Computational protocol: The impact of PPARα activation on whole genome gene expression in human precision cut liver slices

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Protocol publication

[…] Total RNA was purified with RNeasy Minikit columns (Qiagen) and RNA quality was assessed using RNA 6000 Nano chips on the Agilent 2100 Bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands). Purified RNA (100 ng) was labeled with the Ambion WT expression kit (Invitrogen) and hybridized to an Affymetrix Human Gene 1.1 ST array plate (Affymetrix, Santa Clara, CA). Hybridization, washing, and scanning were carried out on an Affymetrix GeneTitan platform according to the instruction by the manufacturer. Arrays were normalized using the Robust Multiarray Average method [, ]. Probe sets were defined according to Dai et al. []. In this method probes are assigned to Entrez IDs as an unique gene identifier. The P-value for the effect of Wy14643 treatment were calculated using an Intensity-Based Moderated T-statistic (IBMT) []. The q-value was calculated as measure of significance for false discovery rate []. The microarray data for the human liver slices were submitted to Gene Expression Omnibus (GSE71731). The microarray data for the human primary hepatocytes have been previously submitted to Gene Expression Omnibus (GSE17251).Gene set enrichment analysis (GSEA) was used to find enriched gene sets in the induced or suppressed genes []. Genes were ranked based on the paired IBMT-statistic and subsequently analyzed for over- or underrepresentation in predefined gene sets derived from Gene Ontology, KEGG, National Cancer Institute, PFAM, Biocarta, Reactome and WikiPathways pathway databases. Only gene sets consisting of more than 15 and fewer than 500 genes were taken into account. Statistical significance of GSEA results was determined using 1000 permutations. […]

Pipeline specifications

Software tools IBMT, GSEA, WikiPathways
Databases GEO Pfam Reactome KEGG BioCarta
Application Gene expression microarray analysis
Organisms Mus musculus, Homo sapiens
Diseases Liver Diseases, Non-alcoholic Fatty Liver Disease