Adenoviral expression of Cre-recombinase was used to delete the RB and/or p53 genes, and retrovirus was used to achieve MYC overexpression.
Growth protocol
Mammary epithelial cells were isolated from 8-week-old female mice and sub-cultured in DMEM/F12 media supplemented with 5% horse serum, 20ng/mL EGF, 10μg/ml insulin, 1ng/ml cholera toxin, 100μg/ml hydrocortisone, 100 U/ml penicillin/streptomycin and 2mM L-glutamine at 37oC in 5% CO2.
Extracted molecule
total RNA
Extraction protocol
RNA was harvested from proliferating cell populations using the RNeasy Blood and Tissue kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's suggested protocols, and quantified on a Nanodrop ND-100 spectrophotometer. RNA quality was assessed by analysis on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
biotin
Label protocol
Labeling was performed by the WT-Ovation Pico RNA amplification system (NuGen Technologies, Inc.)
Hybridization protocol
Arrays were hybridized with fragmented and biotin-labeled target (2.5 µg) in 110 µl of hybridization cocktail. Target denaturation was performed at 99oc for 2 min. and then 45oc for 5 min., followed by hybridization for 18 hrs. Arrays then were washed and stained using GeneChip Fluidic Station 450, and hybridization signals were amplified using antibody amplification with goat IgG (Sigma-Aldrich) and anti-streptavidin biotinylated antibody (Vector Laboratories, Burlingame, CA, USA).
Scan protocol
Arrays were scanned on an Affymetrix GeneChip Scanner 3000, using Command Console Software.
Description
KM-12
Data processing
Raw intensity files from the Affymetrix Mouse Gene 1.0 ST GeneChip arrays were processed using Affymetrix Expression Console version 1.1. Gene-level expression measurements were computed using the iterPLIER algorithm on the core probesets and exported with annotation release 32, dated June 23rd, 2011.
