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Sample GSM364521 Query DataSets for GSM364521
Status Public on Dec 21, 2009
Title PT2-DAPT_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 N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and kept in a humidified 5% CO2 atmosphere at 37°C for the indicated time period (24h).
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 the gamma secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) for 24 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 10.44124614
7896740 12.24876169
7896742 133.6056307
7896744 33.20878419
7896746 304.8141359
7896748 443.3462308
7896750 4.550771031
7896752 427.8736426
7896754 52.5121005
7896756 41.14519343
7896759 102.241564
7896761 122.0877804
7896779 176.8356083
7896798 103.7083323
7896817 122.9121735
7896822 563.0401526
7896859 72.13357392
7896861 17.02778649
7896863 46.81753267
7896865 149.6379193

Total number of rows: 33297

Table truncated, full table size 646 Kbytes.




Supplementary file Size Download File type/resource
GSM364521.CEL.gz 4.2 Mb (ftp)(http) CEL
Processed data included within Sample table

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