An intracranial orthotopic model in severe combined immunodeficiency (SCID) mice was utilized in order to generate infiltrative glioma xenograft tumors. Brains were handled in RNase free conditions, imbedded in OCT compound, frozen immediately and kept at -80°C. Total cellular RNA isolation was carried out for Affymetrix HG-U133 plus2 or Mouse430_2 GeneChip Arrays, according to the manufacturer instructions (Affymetrix, CA)
Growth protocol
Primary GBM glioma stem cells were cultured in NBE medium consisting of Neurobasal A medium with N2 and B27 supplements (Invitrogen, Carlsbad, CA), and human recombinant basic fibroblast growth factor and epidermal growth factor (50 ng/mL each; R&D Systems, Minneapolis, MN). 0923 and 1228A1 (ex vivo culture of 1228 GSCs following one in vivo cycle) GSC lines were used for intracranial mouse glioma models.
Extracted molecule
total RNA
Extraction protocol
RNA extraction was performed via Rneasy
Label
biotin
Label protocol
Standard Affymetrix protocol
Hybridization protocol
Standard Affymetrix protocol
Scan protocol
Standard Affymetrix protocol
Description
sample was taken from a distal area (uninfiltrated) mouse brain, 100% mouse
Data processing
Alternative CDF's were generated by masking probes which showed non-specific hybridization towards mouse in the human arrays and vice versa. Briefly, using raw intensity signals from cel files, individual probes were filtered if they met the following criteria: the net intensity (PM-MM) of each probe pair from the cross-reactive species was less than 10 and greater than 50% of the corresponding signal from human tumor core. Alternatively, to detect cross-reactive human signal on the mouse microarrays, mouse chips were hybridized with pure human cell line RNA and the corresponding same criteria were applied. The remaining probes were grouped into their original probe sets and those that had at least 4 remaining probes were retained in the new chip definition file (cdf). This resulted in alternative cdf’s containing 26,954 probe sets for the HG-U133 plus2 array and 21,675 probe sets for the Mouse430_2 array. The gene expression data were calculated using the MAS5 algorithm following quantile normalization, and the results subjected to statistical analysis using a two-way Anova.
