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 (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 the gamma secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) 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.
