GBM TICs were treated with the antioxidant reagent N-Acetyl-L-Cysteine (NAC) and kept in a humidified 5% CO2 atmosphere at 37°C for the indicated time period (6dd).
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), and 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) and expressed 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. 2009 (PMID 18948646)).
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
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA as detailed in the One-color microarray-based gene expression analysis protocol, v5.5 (www.agilent.com). Dye incorporation and cRNA yield were checked with the NanoDrop.
Hybridization protocol
Labelled cRNAs were hybridized to whole-genome oligonucleotide microarrays (Agilent Technologies) following the manufacturer's instructions (One-color microarray-based gene expression analysis protocol, v5.5). Briefly, 1.5 υg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 100 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 50 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome 4x44K Oligo Microarrays for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent), 1 minute with 37°C GE Wash buffer 2 (Agilent), with the Stabilization solution (Agilent) for 1 minute at room temperature, then dried immediately by rinsing slides into Drying solution (Agilent) for 30 seconds.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G4845A) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm). Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR Lo 10%. Microarray performance was assessed by QC metric tool.
Description
Gene expression analysis. GBM TICs were obtained from tumor samples of patients subjected to surgery at the 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. Cell 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. 2009 (PMID 18948646)).
Data processing
The scanned TIFF images were analyzed with Feature Extraction Software version 10 (Agilent) using default parameters (Agilent protocol GE1-v5_95_Feb07). The output of Feature Extraction was analyzed with the Agi4x44PreProcess package to obtain background subtracted (Agilent parameter gBGSubSignal). Raw data was analyzed using GeneSpring software from Agilent. Normalization of the data was done in GeneSpring using the Quantile normalization.
