Cot 1 human dna

DNA for the array CGH studies was extracted from formalin-fixed, paraffin-embedded tissues with the FormaPure kit from Agencourt. Agilent Sureprint G3 Cancer CGH + SNP 4 × 180k Microarrays were used to identify genome-wide copy number alterations. Briefly, 1µg of tumor and control DNA (normal female gDNA, Corriell Institute) were heated to 95°C for 5 minutes. Random priming was used to label DNA with CY3-dUTP (control) and CY5-dUTP (tumor) dyes from the Agilent SureTag DNA Labeling kit. The labeled DNA was then purified over columns (Agilent) and mixed in equal proportion along with Cot-1 Human DNA (Agilent) for the hybridization steps. To hybridize the DNA to the array, incubation occurred first at 95°C for 3 minutes for denaturation, followed by a 30 minute pre-hybridization step at 37°C and then a hybridization step for 35–40 hours at 65°C. Slides were then washed with Agilent Oligo ArrayCGH wash buffer 1 for 5 minutes at room temperature and wash buffer 2 for 1 minute at 37°C. Upon completion of the washes, slides were scanned using the G2505C Microarray Scanner (Agilent). The data were analyzed using the Agilent CytoGenomics software v 2.0. CGH array data are available at GEO under accession number GSE64765 (super-series GSE64766)..