The IntegraChipTM microarray was designed by the CIT-CGH consortium (Olivier Delattre Laboratory, Curie Institute, Paris; Charles Theillet Laboratory, CRLC Val d'Aurelle, Montpellier; Stanislas du Manoir Laboratory, IGBMC, Strasbourg) and the company IntegraGenTM (http://www.integragen.com). Bacterial artificial chromosome (BAC) clones were chosen to obtain a systematic coverage of the genome, and detailed coverage of regions containing genes previously implicated in carcinogenesis. The median gap between two successive clones is 600 kb. Four replicate spots were deposited on the slides. For each sample, 500-600 ng of sonicated DNA was labelled by Random-Priming (Bioprime Labelling kit, Invitrogen, Carlsberg, CA, USA) according to the suppliers\x{2019} instructions, but for 18h. For tumour DNA, final concentrations of nucleotides were 0.02 mM of dCTP-Cy5 (Amersham Biosciences), 0.1 mM of dATP, dGTP, dTTP and 0.025 mM dCTP. For control DNA, 0.04 dCTP-Cy3 (Amersham Biosciences), 0.2 mM of dATP, dGTP, dTTP and 0.05 mM dCTP. Labelled DNAs were purified using MicroSpin-G50 columns (Amersham Biosciences). COT Human DNA (70 \x{03bc}g, Roche Diagnostics) and Salmon Testes DNA (30 \x{03bc}g, Sigma Chemical Co.) were added to the mixed tumour and reference DNAs. They were subsequently concentrated using a microcon YM-30 (Millipore) and resuspended to obtain 200 \x{03bc}l of hybridization solution (final concentration: 50% formamide, 0.8 x SCC, 10% W/V dextran sulfate, 2% SDS, pH=7). Finally, the probes were pre-annealed for 3 hours at 37°C. Every step was carried out in the HS 4800 hybridization station (Tecan). Slide pre-treatment included rinsing with 2 x SSC, 0.1% SDS at 30 °C for 30s, 2 x SSC at 30 °C for 30s and drying. After probe injection, hybridization was performed for 68h at 37°C under low agitation. Four post-hybridization washes were carried out: 50% formamide, 2 x SSC, 0.1% SDS at 45 °C for 37 s, 4 times; 2 x SSC, 0.1% SDS at 45 °C for 37 sec, 4 times; 4 x SSC, 0.1 Tween 20 at 45 °C for 37 sec, 4 times; slides were finally nitrogen dried and scanned using a ScanArray 4000 (Packard Bioscience). Images were quantified using UCSF Spot 2.1cc (Jain et al., 2002).
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