Tumor specimens were macrodissected to ensure high tumor or normal gland cellularity, respectively, and only sections that contained 70% tumor or non-neoplastic mammary epithelial cells, respectively, were included in the study. Tissue sections were transferred into 300 μl of lysis buffer (NucleoSpin RNA; Macherey&Nagel, Dueren, Germany) and homogenized (Precellyse 24, Bertin Technology, France). mRNA was extracted and purified using a commercial system (NucleoSpin RNA; Macherey&Nagel, Germany).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol (One Cycle Target labeling protocol) from 2ug total RNA.
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45°C on Canine Genome 2.0 Arrays (Affymetrix). GeneChips were washed and stained with standard protocols in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Scanner G3000.
Description
Dog 9
Data processing
Affymetrix CEL files were imported into Partek Genomic Suite Software (Version 6.4, Partek Inc., St. Louis, USA) and processed by the implemented gcRMA workflow (median polish probe set summarization, RMA background correction, quantile normalization).
