Agilent high resolution microarray scanner

The PTEN deletion status in the H1_DL2 and H3 cell lines was studied by DNA copy number analysis. Genomic DNA was extracted from cell lines using the DNAeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany) 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., Grand Island, NY, USA) and labeled using the BioPrime aCGH Genomic Labeling Kit (Invitrogen, Carlsbad, CA, USA) 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, USA) was used as a reference. Labeled DNA was competitively hybridized to SurePrint G3 Human 2 × 400 K CGH microarrays (Agilent Technologies, Santa Clara, CA, USA) following standard Agilent protocols. The slides were scanned at 3-μm 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 (Agilent Technologies) for visualization and analysis. Aberrations were called using the ADM2 algorithm with a threshold setting of 25, centralization on, with a threshold of 25 and an aberration filter min Probes = 5 and min AvgAbsLogRatio = 0.45.

Labelled cDNA was prepared from 1 μg of total RNA using Cy3‐dCTP (GE Healthcare) and SuperScript II reverse transcriptase with random hexamer primers (Invitrogen). Labelled cDNA was purified by Qiagen MinElute column, combined with 10 × CGH blocking agent and 2× Hi‐RPM hybridisation buffer (Agilent) and heated (95°C for 5 min) prior to loading onto microarray slides. Slides were incubated overnight in an Agilent rotating oven at 65°C, 20 rpm. After hybridization, slides were washed (5 min at room temperature) with CGH Wash Buffer 1 (Agilent) and 1 min at 37°C with CGH Wash buffer 2 (Agilent). Slides were scanned immediately, using an Agilent High Resolution Microarray Scanner (Agilent Technologies, Stockport, UK), at 2 μm resolution, 100% PMT. Scanned images were quantified using Feature Extraction software v 10.7.3.1. RNA for tiling microarray hybridization was directly labelled using the Kreatech Universal Linkage System (Leica Microsystems, Milton Keynes, UK), which is based on the stable binding of platinum to the N7 position of guanine, according to the manufacturer's instructions.