gender: female strain: FVB genotype: doxycycline-inducible, bitransgenic MMTV-rtTA/TetO-NeuNT (Her2/Neu) mouse model tissue: normal whole blood cells (blood with plasma fraction removed), at time when breast tumor had reached 1 cm size
Treatment protocol
All mice received doxycycline (2 mg/mL + 5% sucrose) in the drinking water starting at 8 weeks of age. Doxycycline was used to induce tumor development in the bitransgenic mice and breast tumors were allowed to develop to 1.0 cm in diameter after which each tumor-bearing mouse and its corresponding control littermate were euthanized by CO2 inhalation. Tissue samples were collected when the tumors reached ~1 cm size and the samples were snap-frozen in liquid nitrogen.
Growth protocol
All mouse work was performed under IACUC regulations as approved by the Fred Hutchinson Cancer Research Center's animal use committee. For these studies, a doxycycline-inducible, bitransgenic MMTV-rtTA/TetO-NeuNT (Her2/Neu) mouse model was used. Corresponding control mice were transgenic for TetO-NeuNT only and were littermates of the bitransgenic mice. The procedures for animal husbandry and tissue and plasma sample collection were previously reported in Whiteaker J.R., et al., J. Proteome Res. 2007, 10, 3962-3975 and Kelly-Spratt, K.S., et al., J. Proteome Res. 2008, 8, 3613-3618. Briefly, all mice were fed standard laboratory chow (Harland Teklad, 8664) and acidified water ad libitum and kept on a 12 hour light-dark cycle.
Extracted molecule
total RNA
Extraction protocol
The tissues from thymus, spleen, liver, blood cells (blood with plasma fraction removed), and breast were homogenized, and total RNA was isolated using a standard Trizol (Invitrogen, Carlsbad, CA) and chloroform extraction protocol.
Label
biotin
Label protocol
Purified total RNA was normalized to 125ng/ul and reverse transcribed to produce double-stranded cDNA which was amplified, producing biotin-labeled cRNA using the GeneChip HT One-Cycle Target Labeling and Controls Kit (Affymetrix, Santa Clara, CA.)
Hybridization protocol
Biotinylated cRNA (600 ng/ul) was hybridized to GeneChip® Mouse 430 2.0 arrays (Affymetrix, Santa Clara, CA) following the manufacturer’s suggested protocols.
Scan protocol
The arrays were washed and stained using a GeneChip® Fluidics Station and scanned using a GeneChip® Scanner 3000.
Description
Dataset Name: Whole blood array sample name of age- and cage-matched_mouse: TL20070522_3_4_H10 sample ID: 102506243 sample ID of_age- and cage-matched_mouse: 102506240
Data processing
Two sequential quality control checks were implemented to evaluate whether to include results from each of the GeneChips. In the first step, recommendations made by Affymetrix were followed.(Zweig, M. H.; Campbell, G. Clin. Chem. 1993, 39, 561-577) In the second step, we used the affyQCReport and affyPLM software in the Bioconductor package (http://www.bioconductor.org/) to search for poor-quality chips.(Pyeon, D.; et al. Cancer Res. 2007, 67, 4605-4619) In total, 250 chips from both normal and cancer samples from 5 tissues passed these two steps of quality control evaluation. After passing the quality control checks, the DNA microarray data of each separate tissue type were subjected to a quartile normalization step (using Gene Pattern software version 3.0) where intensities of different arrays (but from the same tissue type) were adjusted to the same scale so that the intensity levels from the different arrays could be compared with one another. The normalization was performed on log2 transformed data.
