Agilent genomic dna labeling kit

Array-CGH was performed using an Agilent Sureprint G3 CGH+SNP 4 × 180 K microarray (G4890A; Agilent Technologies) following the manufacturer’s protocols. Briefly, 500 ng of purified DNA of a patient and a control (Agilent Technologies) were double-digested with RsaI and AluI enzymes (Agilent Technologies) for 2 h at 37°C. Each digested sample was labeled for 2 h with the Agilent Genomic DNA Labeling Kit, using Cy5-dUTP for the patient DNA and Cy3-dUTP for the control DNA. Labeled products were purified and prepared according to the Agilent protocol. After probe denaturation and pre-annealing with 50 ng of human Cot-1 DNA (Thermo Fisher Scientific, Yokohama, Japan), hybridization was performed at 65°C for 24 h in a rotating oven. After washing steps, the array slide was scanned with an Agilent Microarray Scanner (G2565CA). The spot intensities were measured and the image files quantified using the Agilent Feature Extraction 11.0.1.1 software. Text outputs from the quantitative analyses were imported into Agilent CytoGenomics 4.0 software (Agilent Technologies).

The method and the CpG island DNA methylation microarray data described here have been deposited in the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) under accession number GSE62383. Briefly, genomic DNA was isolated from tumor tissues by proteinase K digestion followed by phenol/chloroform extraction. After sonication to produce DNA fragments of 200–600 bp, the MethylMiner Methylated DNA Enrichment Kit (Life Technologies, Carlsbad, CA) was used to enrich methylated DNA from one half of the DNA sample while the other half acted as the reference DNA. Enriched and reference DNA fragments were fluorescently labeled using the Agilent Genomic DNA Labeling Kit (Agilent Technologies, Santa Clara, CA), before purification and competitive hybridization using the Agilent DNA Hybridization Kit (Agilent) for 40 h to a 105K rat CpG island microarray (Agilent, Design ID: 021332) covering 96% of regions (approximately 8,000 genes) that are annotated as CpG islands in the rat genome (Nov. 2004, Baylor 3.4/rn4). The fluorescence data extracted from the image of the scanned slides was analyzed using the Agilent Genomic Workbench software (version 6.5).