DNA methylation profiling reveals common signatures of tumorigenesis and defines epigenetic prognostic subtypes of canine Diffuse Large B-cell Lymphoma

In order to assess methylation profiles of dogs affected by DLBCL, a canine CpG microarray platform was developed. Probe design was carried out by the Agilent bioinformatic support team using proprietary prediction algorithms to locate CpG Islands on C. familiaris draft genome as deposited on Ensembl database (CanFam 3.1) and to design high quality oligo-probes. Microarray probes were selected in order to provide the highest possible coverage of dog genome. CDS regions and CpG islands were given top priority. A total of 170,000 probes (60mers, sense orientation) were designed on both CpG and CDS regions. In details, 102,000 probes were designed targeting a total of 36,807 CpG regions while 68,000 probes were directed against 672 CDS; average base pare tiling was 90 bp. Microarray probes were synthesized in situ using the Agilent non-contact ink-jet technology with a 4 x 180K format. Comparative analysis of methylation profiles of lymph nodes from n 37 canine DLBCLc and n 7 control dogs. Enriched-fraction and total gDNA (reference) obtained from 44 lymph node samples (37 cDLBCLs and 7 control dogs) were labelled independently with cyanine 5-deoxyuridine triphosphate (dUTP) and cyanine 3-dUTP, respectively. Sample labeling was performed by using SureTag Complete DNA Labeling Kit (Agilent Technologies) following the manufacturer’s recommendations.