The isolation and culture of multipotential human central nervous system progenitor cells has been described previously (Messam et al., 2003). Briefly, cells were grown as two-dimensional attached cultures on poly-D-lysine (PDL) coated plasticware at 37°C in 5% CO2. Progenitor cells were grown in serum-free neurobasal medium supplemented with N2 components, neural survival factor, 2mM L-glutamine, 50 ug/mL gentamycin, recombinant basic fibroblast growth factor (25ng/ml bFGF; Peprotech), and recombinant epidermal growth factor (20ng/ml EGF; Peprotech). To induce differentiation, progenitor growth media was removed, cells were washed twice with 37°C phosphate buffered saline, and PDA media was added. PDAs were maintained in minimal essential medium (Cellgro) supplemented with 10% FBS, 2 mM L-glutamine and 50 mg/mL gentamycin. For differentiation experiments cells were plated 3-4 days prior to isolation of RNA/protein. All time points up to and including 3 days post-differentiation were plated 3 days prior to differentiation. Later time points were differentiated in flasks, and plated 3-4 days prior to isolation of RNA/protein. Cells were plated at a density of 2x105 cells per well in 6-well plates for RNA isolation and plated on PDL coated glass cover slips in 12-well plates at a density of 5x104 cells per well for immunofluorescence.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted by sample using the Qiagen RNAeasy kit. Post extreaction, RNA quality and quantity was ensured using the Bioanalyzer (Agilent, Inc) and NanoDrop (Thermo Scientific, Inc) respectively.
Label
Biotin
Label protocol
200 nanograms of total RNA per sample was used in conjunction with the Affymetrix recommended protocol for the GeneChip Human Gene 1.0 ST Array (Affymetrix, Inc).
Hybridization protocol
Labeled cDNA was hybridized, washed and stained to separate Affymetrix GeneChip Human Gene 1.0 ST Arrays by sample according to the protocols described by Affymetrix (Affymetrix, Inc). Per staining, streptavidin phycoerythrin solution was used (Molecular Probes, Carlsbad, CA) and enhanced by an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA).
Scan protocol
The Affymetrix Gene Chip Scanner 3000 was used to scan each Affymetrix GeneChip Human Gene 1.0 ST Array. After, gene probe intensities were generated using the Affymetrix AGCC software (Affymetrix, Inc).
Description
Human Fetal Brain NPC 0h Replicate 3
Data processing
The Affymetrix Expression Console (Affymetrix, Inc) was used to summarize the gene probe intensities and generate 33,297 RMA normalized gene fragment expression values for each hybridized cRNA.
