breed: German-Shepherd-Mix age in years: 13 histologic grade: Grade III tumor size (cm): 7 affected lymph node: none (no lymph node metastasis) postoperative survival (months): >24 era: negative her-2: +
Extracted molecule
total RNA
Extraction protocol
Tissue specimens were snap frozen in liquid nitrogen within 15 minutes after resection and stored at -80ºC until further use. Macrodissection was performed on all tumor specimens to ensure high tumor cellularity. Only sections with more than 70% carcinoma cells were included in the study as shown by digital image analysis (Scanscope T3, Aperio, Vista, USA; Zeiss Axiovision, Jena, Germany). For mRNA isolation, tissue sections were transferred into 300 µl of lysis buffer (NucleoSpin RNA XS; Macherey&Nagel, Dueren, Germany) and homogenized (Precellyse 24, Bertin Technology, France). RNA was extracted and purified using a commercial system (NucleoSpin RNA XS; Macherey&Nagel, Düren, Germany). The RNA quality was controlled using the BioAnalyzer (Agilent Technologies, USA) and only high quality RNA (RIN>8) was used for microarray analyses. Affymetrix GeneChip hybridization (Canine Genome 2.0 Array) was performed with 2 µg total RNA according to the manufacturer’s recommendations.
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
n/a
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).
