Computational protocol: Sex differences in microRNA-mRNA networks: examination of novel epigenetic programming mechanisms in the sexually dimorphic neonatal hypothalamus

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Protocol publication

[…] Total RNA was extracted from the PN2 hypothalamus using TRIzol reagent (Invitrogen), and total RNA was isolated according to the manufacturer’s protocol. One microgram each from two littermates was pooled (pooled samples: F/Veh n = 5, M/Form n = 7, F/Veh n = 6) and submitted for microarray analysis (Affymetrix GeneChip miRNA 3.0 Array). Microarray analyses were performed as described previously with minor modifications []. R (version 2.14.2) was used with the limma package to generate gene expression values and to fit linear models with the predictor value of sex to these data [, ]. Based on the observation that in microarray experiments unexpressed transcripts can be most reliably detected by their low variability across all samples, miRNAs with total variance values in the lowest 0.25 quantile were excluded []. Thresholds for multiple comparisons in determining sex-biased miRNAs were set at a false discovery rate (FDR) ≤ 0.05. Multivariate analyses of miRNA expression were performed using SIMCA-P software (UMETRICS). [...] miRNA annotations were derived from miRBase v.21 []. miRNA cluster assignments were obtained from MetaMirCluster []. Transcription start site (TSS) mapping data was produced by the FANTOM5 Consortium and obtained as a UCSC Genome Browser public track (FANTOM5 TSS peaks [robust]) []. Ago footprint annotations and alignment were derived from RefSeq mRNA []. Genomic coordinates reference Mus musculus genome assembly MGSCv37 (mm9) []. The network of Ago HITS-CLIP connections was generated using Cytoscape v.3.1.1 []. Clustering of enriched Gene Ontology terms (GO Biological Processes release 03/20/2014) was performed with the Cytoscape plug-in ClueGo v.2.1.6 [, ]. Previously validated miRNA-target interactions were accessed through the database DIANA-Tarbase v7.0 []. […]

Pipeline specifications

Software tools limma, ClueGO
Databases DIANA-TarBase miRBase FANTOM UCSC Genome Browser
Applications miRNA array analysis, Genome data visualization
Diseases Hypothalamic Neoplasms, Disorders of Sex Development
Chemicals Estrogens