Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression analysis of hippocampal tissue from epilepsy surgery

ABSTRACT: Samples used in the study originated from three UK sites: the Walton Centre for Neurology and Neurosurgery in Liverpool, the Salford Royal Hospital in Salford and the Southern General Hospital in Glasgow. We recruited patients with pharmacoresistant mesial temporal lobe epilepsy for whom a therapeutic temporal lobectomy was being undertaken. After surgery, the hippocampus was divided into two portions: (1) one portion was preserved for RNA isolation, and (2) the other portion underwent histological analysis by an experienced neuropathologist. Frozen post-mortem histologically-normal hippocampal samples from donors with no known brain diseases were obtained from the MRC Edinburgh Brain Bank (Edinburgh, UK) and the Queen Square Brain Bank (London, UK). Brain samples were disrupted and homogenized in an appropriate volume of QIAzol lysis reagent (Qiagen, Crawley, United Kingdom) by using a TissueRuptor handheld rotor-stator homogenizer (Qiagen, Crawley, United Kingdom). Total RNA was extracted from the homogenates using the RNeasy Lipid Tissue Mini Kit (Qiagen, Crawley, United Kingdom), according to the manufacturer’s instructions. RNA quality was examined by capillary electrophoresis on an Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA) and Agilent 2100 Expert software was used to calculate the RNA Integrity number (RIN) of each sample. Purity of the RNA sample was assessed using a NanoDrop1000 Spectrophotometer. Capillary electrophoresis traces were also examined. Samples with RNA integrity number scores (RIN) below 6, obvious RNA degradation, significant 18S or 28S ribosomal RNA degradation, ratio of absorbance at 260nm and 280nm <1.95, or with noticeable DNA or background contaminants did not pass QC, and were withheld from microarray analysis. The microarrays were processed at the Centre for Genomics Research in the University of Liverpool (http://www.liv.ac.uk/genomic-research/). 50ng of total RNA was amplified and labelled using the Agilent Low Input Quick Amp One-Colour Labeling Kit and labelled RNA was hybridized to Agilent SurePrint G3 Custom Exon 8x60K Microarrays designed to contain probes for each exon of 936 selected genes, including all known SLC genes. Standard Agilent protocols were followed. One array failed on five of the QC criteria and, hence, was excluded. Intensity data were extracted from the remaining arrays using the Feature Extraction Software, in line with the manufacturer’s recommendations. The uploaded files contain data both for a custom exon array designed to contain probes for each exon of 936 selected genes and for a custom gene expression array designed to contain gene expression probes for genes across the whole genome.

ORGANISM(S): Homo sapiens

SUBMITTER: Nasir Mirza

PROVIDER: E-MTAB-3123 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The objective was to characterize differences in the secretome of human omental compared with subcutaneous adipose tissue using global gene expression profiling. Gene expression was measured using Affymetrix microarrays in subcutaneous and omental adipose tissue (n=3 independent subjects; 6 arrays). Predictive bioinformatic algorithms were employed to identify those differentially expressed genes that code for secreted proteins and to identify common pathways between these proteins. All patients provided informed written consent before inclusion in the study which was approved by the North of Scotland Research Ethics Committee (NOSREC). The subcutaneous and omental white adipose tissue biopsies were analysed from three obese patients (BMI 39.1+/-1.9 kg/m2; age 36 +/- 8 years) who had fasted overnight. Subcutaneous and omental white adipose tissue biopsies were taken at the time of surgery for vertical banded gastroplasty from n=3 independent subjects who had fasted overnight. (6 arrays). Adipose tissue samples were obtained within 5 minutes of the tissue being extracted from the patients and frozen immediately in liquid nitrogen. Subjects had fasted overnight prior to surgery. Total RNA was extracted using the RNeasy Lipid tissue mini kit followed by incubation with RNase free DNase (Qiagen, Crawley, West Sussex) for DNA-free RNA, following the manufacturer's instructions (Qiagen, Crawley, West Sussex). RNA was quantified on the Agilent 2100 Bioanalyser (Agilent Technologies, South Queensferry, West Lothian). This also shows the quality of the RNA extracted from the tissues. Only RNA showing no degradation (RIN greater than 9.0) was processed further. The DNA-free RNA from the subcutaneous and omental adipose tissue samples from the patients was then sent to a commercial company for analyses. The RNA from three volunteers (6 arrays ) was fragmented and hybridised to Human Affymetrix U133 plus 2 (Santa Clara, CA) gene chips following the Affymetrix protocol by the MRC Geneservice (MRC HGMP Resource centre, Babraham, Cambridge). Briefly, the Affymetrix one-cycle target labelling and control reagents were used to synthesize Biotin-labelled cRNA. The concentration of the cRNA was measured using a Nanodrop (Thermoscientific) and Agilent bioanalyser to rule out the possibility that only very short cRNA products were formed. This could have caused a 3’-5’ bias and influenced the data analysis. Approximately 20µg cRNA was used for further fragmentation and finally 10µg was used for the hybridisations. The Affymetrix protocols were followed for the hybridisations, washing, staining and scanning of the chips

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Gene expression analysis of hippocampal tissue from epilepsy surgery

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