Differentiation medium, DMEM,F12 supplemented with 50μg/ml apo-Transferrin, 5ng/ml sodium selenite, 100μM Putrescine, 130nM Progesterone, 200 nM T3, 1% HS, 1% Pennicilin/Streptomycin, 2mM glutamine, 0.1% Sodium bicarbonate and 15mM Hepes. Chemical treatments for micro-array analysis
Growth protocol
Oli-Neu cell line were maintained in DMEMF12 (Invitrogen # 21331) supplemented with 50μg/ml apo-1 Transferrin (Sigma T-1147), 5ng/ml sodium selenite (Sigma S-9133), 5μg/ml Insulin (Sigma I-0516),100μM Putrescine (Sigma P-7505), 22nM Progesterone (Sigma P-7556), 0.5 nM T3 (Sigma T-6397), 1% HS (Invitrogen 26050), 1% nicillin/Streptomycin (Invitrogen 15070), 2mM glutamine Invitrogen 25030), 0.1% Sodium bicarbonate (Invitrogen 25080) and 15mM Hepes Invitrogen 15630)
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
The One-Cycle Eukaryotic Target Labeling Assay (One-Cycle Target Labeling and Control Reagents,Affymetrix, P/N 900493) was used to produce fragmented biotinylated cRNA from total RNA as described in the standard Affymetrix protocol (P/N 701021 Rev. 5).
Hybridization protocol
In our study, 3 μg of total RNA isolated were first reverse tanscribed using a T7-Oligo(dT) Promoter Primer in the first-strand cDNA synthesis reaction. Following RNase H-mediated second-strand cDNA synthesis, the double-stranded cDNA was purified and served as a template in the subsequent in vitro transcription (IVT) reaction. The IVT reaction was carried out in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling. The biotinylated cRNA targets were then cleaned up, an aliquot of 20 μg was fragmented, and finally 15 μg were hybridized to GeneChip® Mouse Genome 430 2.0 Array (Affymetrix, P/N 900495, 900496, 900497). Chips were washed andlabeled in the fluidics station, using protocol EuKGE-WS2v_45 After scanning.
Scan protocol
Affymetrix GeneChip® Scanner 3000 7G
Description
Oli-neu after 10 h treated with Forskolin in Experiment 2
