Peripheral blood samples were collected in EDTA containing tubes and diluted 1:6 in Erythrocyte lysis buffer (0.13 M NH4CL, 0.1 M KHC03 and 1.2x10-4 M EDTA ) in order to eliminate red blood cells. After incubation for 30 minutes at 4ºC samples were centrifuged at 1500 rpm for 15 minutes and the supernatant was discharged in order to obtain a pellet composed mainly by peripheral blood leukocytes (PBL). The procedure was repeated until a clear, white pellet was obtained. The two canine mammary carcinoma cell lines, CMM115 and CMM26, were cultivated in RPMI1640 medium supplemented with 10% bovine fetal serum. The cells were incubated in 75 cm3 tissue culture flasks, with air containing 5 % CO2 at 37oC. After the formation of a confluent monolayer, cells were harvested by washing once with PBS, pH 7,3 and incubation with in PBS containing 0,02 % EDTA and 0,25 % trypsin for five minutes a 37°C. RNA isolation from the PBL pellets and cell lines was performed using a commercial kit (Nucleo Spin RNAII, Macherey-Nagel) according to the manufacturer’s instructions with subsequent DNAse treatment.
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
Data processing
Affymetrix CEL files were imported into Partek Genomic Suite Software (Version 6.5, Partek Inc., St. Louis, USA) and processed by the implemented RMA workflow (median polish probe set summarization, RMA background correction, quantile normalization).