Affymetrix Generic Hybridization
nucleic acid labeling protocol
P-AFFY-2 Affymetrix biotin labelling
Background correction,quantile normalization, and gene expression analysis were performed using RMA (Bolstad, B.M., Irizarry R. A., Astrand, M., and Speed, T.P. (2003)A Comparison of Normalization Methods for High Density Oligonucleotide Array Data Based on Bias and Variance. Bioinformatics 19(2):185-193).
Following manufacturers instructions (Parameters: Scanning hardware = GeneChip Scanner 3000 [Affymetrix], Scanning software = MAS/GCOS/GREX [Affymetrix])
siRNA silencing of GABPA or GAPDH control in MCF10A cells performed with three biological repeats. MCF10A cells were maintained in DMEM/F12 containing 5% horse serum, 20 ng/ml EGF, 10 ug/ml insulin, 100 ng/ml cholera toxin and 0.5 ug/ml hydrocortisone. Cells were plated out into 6-well plates (520,000/well) into a mixture of 83% growth medium (without EGF), 17% OptiMEM (Invitrogen), 20 nM siRNA and 0.33% Lipofectamine2000 (Invitrogen) followed by replacement of the transfection mix with EGF-depleted growth media after 12 hours. The siRNA constructs were ON-TARGETplus SMART pools from Dharmacon for GABPA and GAPDH (control). Forty eight hours post-seeding, RNA was isolated
RNA was isolated using the RNeasy kit (QIAgene) according to the manufacturerﾒs protocol and quantified with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies). (Parameters: Extracted product = total_RNA, Amplification = none)