Computational protocol: Maternal Western-Style High Fat Diet Induces Sex-Specific Physiological and Molecular Changes in Two-Week-Old Mouse Offspring

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Protocol publication

[…] Per offspring liver sample, 100 ng of purified RNA was used for the preparation of labelled cDNA, applying the Ambion Whole Transcript (WT) Expression kit (Life Technologies, Carlsbad, USA) in combination with the Affymetrix GeneChip WT Terminal Labelling kit (Affymetrix, Santa Clara, USA). All samples were hybridized at one time point to Affymetrix GeneChip Mouse Gene 1.1 ST arrays according to standard Affymetrix protocols. Quality control and normalisation were performed using Bioconductor software packages integrated in an on-line pipeline . Normalised expression estimates of probe sets were computed by the robust multiarray (RMA) analysis algorithm available in the Bioconductor library AffyPLM using default settings . Probe sets were redefined according to Dai et al. and assigned to unique gene identifiers (IDs) of the Entrez Gene database, resulting in 21,225 assigned Entrez IDs. Array data were submitted to the Gene Expression Omnibus and are available under accession number GSE46359. [...] Of the 21,225 defined genes covered by the microarray, only genes with an intensity value of ≥20 on at least 4 arrays and an interquartile range (IQR)≥0.1 were selected for further analysis. The remaining 11,362 genes were ranked based on their IQR value, and principal component analysis (PCA) was applied on the top 1,000 most variable genes using MultiExperimentViewer, version 4.8.1 , . Signal 2log ratios, which represent fold changes (FC), and related significances of change were calculated from the mean signal intensities of maternal WSD groups and maternal LFD groups using intensity based-moderated t-statistics (IBMT) implementing empirical Bayes correction . Resulting 2log ratios and p-values were applied for further descriptive bioinformatic analysis of the data. The method description and results of quantitative real-time PCRs that confirm the microarray data are presented as Supporting Information ( and ).Ingenuity Pathway Analysis (IPA, Ingenuity® Systems, was used to relate the microarray data to biological functions and canonical pathways. Genes that significantly changed in expression due to maternal diet (p-value≤0.01) were subjected to this comprehensive pathway and network analysis.Gene sets related to biological functions that were most significant to the data, were expressed in heat maps using MultiExperimentViewer, version 4.8.1 , . By hierarchical clustering based on Pearson correlation, gene data were organized into binary trees that group similar elements together. […]

Pipeline specifications

Software tools affyPLM, IBMT, IPA
Databases GEO Gene
Application Gene expression microarray analysis
Organisms Mus musculus
Chemicals Cholesterol