Computational protocol: ICAD Deficiency in Human Colon Cancer and Predisposition to Colon Tumorigenesis: Linkage to Apoptosis Resistance and Genomic Instability

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Protocol publication

[…] The profile of chromosome aberrations in colonic tissue (isolated from paraffin-embedded tissues) from DMH-treated WT or ICAD−/− mice was assessed using an oligo microarray-based CGH with a chip containing 105,000 mouse genomic probes (Agilent, Santa Clara, CA; www.agilent.com). The reference controls were DNA isolated from colon tissue of age-matched naïve WT or ICAD−/− mice. The reference and test DNAs were digested with Alu I and Rsa I (Promega, Madison, WI), and purified with the QIAprep Spin Miniprep kit (QIAGEN, Germantown, MD). Test DNA (1 µg) and reference DNA (1 µg) were labeled by random priming with Cy5-dUTP and Cy3-dUTP, respectively, using the Agilent Genomic DNA Labeling Kit Plus. The individually labeled test and reference samples were concentrated using Microcon YM-30 filters (Millipore, Billerica, MA) and then combined. Following probe denaturation and pre-annealing with mouse Cot-1 DNA, hybridization was performed at 65°C with rotation for 40 h at 20 rpm. Four steps were done with Agilent Oligo CGH washes: Wash buffer 1 at room temperature for 5 min, wash buffer 2 at 37°C for 1 min, an acetonitrile rinse at room temperature for 1 min and a 30 sec wash at room temperature in Agilent's Stabilization and Drying Solution. All slides were scanned on an Agilent DNA microarray scanner. Data including Copy Number Variations were obtained by Agilent Feature Extraction software 9 and analyzed with Agilent Genomic Workbench software 5.0, using the statistical algorithms z score and ADM-2 according to sensitivity threshold respectively at 2.5 and 6.0 and a moving average window of 0.2 Mb. […]

Pipeline specifications

Software tools Agilent Feature Extraction, Agilent Genomic Workbench
Application aCGH data analysis
Organisms Mus musculus, Homo sapiens
Diseases Colonic Neoplasms, Deficiency Diseases, Neoplasms