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Sample GSM364531 Query DataSets for GSM364531
Status Public on Dec 21, 2009
Title PT3-LLNLe_24h
Sample type RNA
 
Source name Human glioblastoma (GBM) tumor initiating cells (TICs).
Organism Homo sapiens
Characteristics GBM TICs were obtained from tumor samples of patients subjected to surgery at Neurosurgery Department of the San Martino Hospital in Genova. An informed consent was obtained from all patients before surgery as required by the Ethic Board.
Cells isolation was described in detail elsewhere (SOX2 Silencing in Glioblastoma Tumor Initiating Cells Causes Stop of Proliferation and Loss of Tumorigenicity.
Gangemi RM, Griffero F, Marubbi D, Perera M, Capra MC, Malatesta P, Ravetti GL, Zona GL, Daga A, Corte G. Stem Cells. 2008 Nov 6)
Biomaterial provider GBM TIC cells were provided from Dr. A. Daga (IST, Genova, Italy).
Treatment protocol GBM TICs were treated with the gamma secretase inhibitor z-Leu-leu-Nle-CHO (LLNle) and kept in a humidified 5% CO2 atmosphere at 37°C for the indicated time period (48h).
Growth protocol Proliferation medium was DMEM-F12/Neurobasal additioned with 1% v/v B27 supplement, (Gibco Ltd, Paisley, Scotland), 2 mM L-glutamine (Gibco Ltd), recombinant human fibroblast growth factor (FGF-2, 10 ng/ml Peprotech, London, UK), recombinant human epidermal growth factor (EGF, 20 ng/ml Peprotech). The medium was changed twice a week. Under these conditions, TICs were grown as a monolayer in flasks coated with Matrigel (BD Biosciences, San Jose, CA) express stem cell markers, maintained intact self-renewal capacity, partial multilineage differentiation ability and gave rise to tumor when injected orthotopically in nude mice (SOX2 Silencing in Glioblastoma Tumor Initiating Cells Causes Stop of Proliferation and Loss of Tumorigenicity.
Gangemi RM, Griffero F, Marubbi D, Perera M, Capra MC, Malatesta P, Ravetti GL, Zona GL, Daga A, Corte G. Stem Cells. 2008 Nov 6).
Extracted molecule total RNA
Extraction protocol miRNeasy Mini Kit (Qiagen) with DNase treatment.
RNA concentration and purity were determined from measuring absorbance at 260 and 280 nm; 2 μg total RNA was run on a 1% denaturing gel and 100 ng were loaded on the 2100 Bioanalyzer (Agilent, Palo Alto, CA) to verify RNA integrity.
Label Biotin
Label protocol Briefly, accordingly to the recommendations of the manufacturer, 100 ng total RNA was used in the first-round synthesis of double-stranded cDNA. The RNA was reverse transcribed using a WT cDNA synthesis and amplification kit (Affymetrix). The resulting biotin-labeled cRNA was purified using an IVT clean-up kit (Affymetrix) and quantified using a UV spectrophotometer (A260/280; Beckman, Palo Alto, CA). An aliquot (15 µg) of cRNA was fragmented by heat and ion-mediated hydrolysis at 94°C for 35 minutes.
 
Hybridization protocol Fragmented cRNA, run on the Bioanalyzer to verify the correct electropherogram, was hybridized in a hybridization oven (16 hours, 45°C) to a Human Gene 1.0 ST array (Affymetrix) representing whole-transcript coverage. The washing and staining of the arrays with phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, OR) was completed in Fluidics Station 450 (Affymetrix).
Scan protocol The arrays were subsequently scanned using a confocal laser GeneChip Scanner 3000 7G and GeneChip Command Console (Affymetrix).
Description Gene expression data from GBM TICs were treated with the gamma secretase inhibitor z-Leu-leu-Nle-CHO (LLNle) for 48 hours.
Data processing Affymetrix raw data files (CEL files) are used as input files in expression console environment (Affymetrix, Inc., Santa Clara, CA). Briefly, Cel files were processed using Robust Multi-Array Analysis procedure (Irizarry et al., 2003). The RMA method was used, first, to convert the intensities from the multiple probes in a probeset into a single expression value with greater precision and reduced background noise, relying on the perfect match probes only (ignoring the mismatch probes) and, after, to normalize by sketch quantile normalization. Also quality assessement were performed in expression console environment.
 
Submission date Jan 26, 2009
Last update date Dec 21, 2009
Contact name Massimiliano Monticone
E-mail(s) massimiliano.monticone@istge.it
Organization name IRCCS AOU San Martino – IST, Genova
Lab S.S. Biofisica e Citometria
Street address Largo R. Benzi, 10
City Genova (GE)
ZIP/Postal code 16132
Country Italy
 
Platform ID GPL6244
Series (1)
GSE14581 The gamma secretase inhibitor LLNle induces apoptosis of human GBM tumor initiating cells

Data table header descriptions
ID_REF
VALUE absolute expression value

Data table
ID_REF VALUE
7896738 7.960759068
7896740 11.63894187
7896742 157.3992657
7896744 43.16460855
7896746 192.9345889
7896748 95.23143717
7896750 3.758869946
7896752 271.4568351
7896754 44.04157018
7896756 49.83906161
7896759 103.3597016
7896761 88.8069081
7896779 135.3994436
7896798 98.81478406
7896817 111.5278341
7896822 156.0554574
7896859 80.03099828
7896861 11.75164651
7896863 46.51596334
7896865 158.1260227

Total number of rows: 33297

Table truncated, full table size 646 Kbytes.




Supplementary file Size Download File type/resource
GSM364531.CEL.gz 4.2 Mb (ftp)(http) CEL
Processed data included within Sample table

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