Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene (mRNA) expression profiling in frontal cortex of ethanol-treated mice

ABSTRACT: Analysis of early stages of alcohol dependence at the gene expression level. The hypothesis tested in the present study was that ethanol-treatment impact gene expression in a mouse model of high ethanol consumption. Results provide important information of genes and pathways being affected by ethanol actions in the mouse frontal cortex. Total RNA obtained from frontal cortex from mice treated with 20% ethanol solution for 20 days. Control group is composed of untreated animals. Frontal cortex (FCtx) tissue was dissected to produce a 2-mm coronal section from the most rostral portion of the mouse brain devoid of olfactory bulbs (coordinates Bregma +1.56 to +3.56). The dorsal part of this coronal section, cut immediately above the forceps minor of the corpus callosum as the anatomical landmark, was used for RNA extraction. This section of the cortex is mostly composed of frontal associated cortex (FrA), cingulate cortex area 1 (Cg1), prelimbic cortex (PrL), and primary (M1) and secondary (M2) motor cortices, as depicted in the mouse brain atlas (Franklin and Paxinos 2007). Samples from both alcohol-treated and control groups were always included in each batch of extracted RNA. Total RNA was extracted using the mirVanaM-BM-. miRNA Isolation kit (Ambion, Austin, TX) according to the manufacturerM-bM-^@M-^Ys instructions. Yield and quality of the total RNA preparation was determined using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). For mRNA expression profiling, biotin-labeled cRNA was prepared using Illumina TotalPrep RNA Amplification kit (Ambion, Austin, TX) and then hybridized to Illumina MouseRef-8 v2.0 Expression BeadChips (Illumina, San Diego, CA). The quality of the Illumina bead summary data was assessed using the Bioconductor packages Lumi and arrayQualityMetrics. Data preprocessing included variance stabilization and quantile normalization using the Lumi package. Statistical analysis comparing ethanol-treated and control groups was performed using the Bioconductor package limma, which implements an empirical Bayes approach in R (Smyth 2005). False discovery rate (FDR) was assessed using the Benjamini-Hochberg method.

ORGANISM(S): Mus musculus

SUBMITTER: Yury Nunez

PROVIDER: E-GEOD-50427 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Comparison of gene expression in labouring and non-labouring myometrium. In this work, gene expression in 48 (22 labouring, 26 non-labouring) myometrial samples was measured using Illumina HT-12 v4.0 BeadChips. Biopsy samples of full thickness lower segment specimens of human myometrium were selected from the Edinburgh Reproductive Tissue Biobank (ERTBB, http://www.crh.ed.ac.uk/biobank/). All tissue samples were collected during Caesarean section from participant tissue donors with informed and written consent, according to the ethical approval and governance granted to the Edinburgh Reproductive Tissues BioBank by the West of Scotland Research Ethics Committee 4 (09/S0704/3). In order to maximise biological replicates on the array, all 22 labouring samples that fulfilled our inclusion criteria and that were stored in the ERTBB as of August 2012 were selected. In order to minimise inter-group variation in baseline characteristics, a second group of non-labouring samples (n=26) were selected by matching to labouring samples on maternal BMI, gestational age, parity and maternal age. We did not exclude smokers or women who had received medication to induce labour. We restricted the number of non-labouring samples to 26 so that all samples (n=48) could fit on three array chips. Forty-five of the samples had been transferred into RNAlater® (Life Technologies, Invitrogen, Carlsbad, CA, USA) immediately after collection and then stored at -80°C. Three labouring samples had not been transferred to RNAlater® before freezing but were also included in the array analysis in order to maximise the number of labouring samples. Total RNA was isolated using TRI Reagent® and the Qiagen RNeasy Lipid Tissue kit protocol (Qiagen, Valencia, CA, USA). The quantity and quality of RNA was assessed using a NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA). RNA quality was further assessed in the biotin-labelled samples by the Wellcome Trust Clinical Research Facility, Edinburgh using a Bioanalyzer 2100 (Agilent Technologies, Wilmington, DE, USA). To prepare samples for the microarray experiment, 450ng total RNA was amplified and biotin-labelled using the Illumina® TotalPrepTM RNA Amplification Kit (Ambion, Austin, TX, USA). Samples were split randomly over four Illumina HT-12 v4.0 BeadChips to minimise any effect of inter-chip variability. One sample was used per well. The chips were imaged using a BeadArray Reader and raw data was obtained with Illumina BeadStudio software. Raw data were preprocessed using the Lumi package in R version 2.14.1. The data was subjected to Robust Multichip Average (RMA) background correction before quantile normalisation to remove non-biological systematic variation.

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Gene (mRNA) expression profiling in frontal cortex of ethanol-treated mice

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