SurePrint G3 Human CGH Microarray Kit, 2x400K

Protocol tips

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	Do not use DEPC-treated water, only water provided in the kit or nuclease-free water

Protocol tips
Do not use DEPC-treated water, only water provided in the kit or nuclease-free water

Publication protocol

aCGH analysis
Genomic DNA was extracted from cell lines using the DNAeasy Blood and Tissue Kit (Qiagen) following the manufacturer's instructions. DNA was eluted in water, fragmented to an average size of 200–500 bp using DNAse1 (rDNAse1, Ambion, Life Technology Ltd., Paisley, UK) and labeled using the BioPrime aCGH Genomic labeling Kit (Invitrogen, Paisley, UK) and Cy3 and Cy5 dyes purchased from GE Healthcare (Chalfont St. Giles, UK) following standard protocols for Agilent aCGH. Commercially available female DNA pooled from multiple anonymous donors (Promega, Madison, WI) was used as a reference for each of the aCGH experiments. Labeled DNA was competitively hybridized to SurePrint G3 Human 2 × 400 k CGH microarrays (Agilent Technologies, Santa Clara, CA) following standard Agilent protocols. The slides were scanned at 3 m resolution using the Agilent High-Resolution Microarray scanner and the image data were extracted using Feature Extraction (Agilent Technologies). FE extraction files were imported into Genomic Workbench 7.0 (Agilent Technologies) for visualization and analysis. Aberrations were called using the Aberration Detection Method 2 (ADM2) algorithm with a threshold setting of 25, centralization on with threshold of 25, and an aberration filter min Probes = 3 and minAvgAbsLogRatio = 0.25. The ADM-2 algorithm identifies all aberrant intervals in a given sample with consistently high or low log ratios based on the statistical score that represents the deviation of the average of the log ratios from the expected value of zero. Hierarchical clustering was done by using the clustering analysis implemented in the Genomic Workbench software. This is based on Euclidian distance calculations using aberration scores.



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Manufacturer protocol

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