DNA was isolated using the QiAmp DNA isolation kit (Qiagen, Hilden, Germany) and subsequently quantified by a fluorometric assay (Quant-iT Pico Green dsDNA Kit, Invitrogen, Karlsruhe, Germany). Fluorescence was measured using a TECAN (Salzburg, Austria) SpectrafluorPLUS microplate fluorescence reader with an excitation wavelength at 485 nm and an emission wavelength at 535 nm. DNA concentrations were calculated from a standard curve of double-stranded control DNA provided with the kit that was measured in triplicate at the concentrations 30, 3, 0.3, and 0.03 ng/µl (measured by fluorometry).A volume of 2.5 µl out of 4 ng/µl dilutions from cell lines and control DNA was used as starting material for the amplification. The phi29-amplification was carried out according to the Repli-G kit manufacturer's instructions (Qiagen) using an incubation time of 16 h. Repli-G reactions were checked by a real time based intra Alu-PCR assay. Additionally DNA concentration was fluorimetrically measured with Quant-iT Pico Green dsDNA Kit (Invitrogen) and ranged between 10-30 Ž¼g.
Label
cy3
Label protocol
10 µg of amplified and purified DNA were labeled using the BioPrime Array CGH Genomic Labeling kit (Invitrogen) according to the manufacturer¢â¬â„¢s instructions in a volume of 50 µl with a modified dNTP mix containing 120 µM each of dATP, dGTP, and dCTP; 60 µM dTTP; and 60 µM Cy5-dUTP or Cy3-dUTP. Labeled DNA was purified using QIAquick PCR purification columns (Qiagen). Tumor and reference DNA were pooled, mixed with 50 µg Cot-1 DNA (Invitrogen) and concentrated to a volume of 20 µl in a vacuum centrifuge. DNA was then mixed with an equal amount of 2x formamide buffer, denaturated at 95°C for 5 min and preincubated at 37°C for 30 min in a waterbath.
DNA was isolated using the QiAmp DNA isolation kit (Qiagen, Hilden, Germany) and subsequently quantified by a fluorometric assay (Quant-iT Pico Green dsDNA Kit, Invitrogen, Karlsruhe, Germany). Fluorescence was measured using a TECAN (Salzburg, Austria) SpectrafluorPLUS microplate fluorescence reader with an excitation wavelength at 485 nm and an emission wavelength at 535 nm. DNA concentrations were calculated from a standard curve of double-stranded control DNA provided with the kit that was measured in triplicate at the concentrations 30, 3, 0.3, and 0.03 ng/µl (measured by fluorometry).A volume of 2.5 µl out of 4 ng/µl dilutions from cell lines and control DNA was used as starting material for the amplification. The phi29-amplification was carried out according to the Repli-G kit manufacturer's instructions (Qiagen) using an incubation time of 16 h. Repli-G reactions were checked by a real time based intra Alu-PCR assay. Additionally DNA concentration was fluorimetrically measured with Quant-iT Pico Green dsDNA Kit (Invitrogen) and ranged between 10-30 Ž¼g.
Label
cy5
Label protocol
10 µg of amplified and purified DNA were labeled using the BioPrime Array CGH Genomic Labeling kit (Invitrogen) according to the manufacturer¢â¬â„¢s instructions in a volume of 50 µl with a modified dNTP mix containing 120 µM each of dATP, dGTP, and dCTP; 60 µM dTTP; and 60 µM Cy5-dUTP or Cy3-dUTP. Labeled DNA was purified using QIAquick PCR purification columns (Qiagen). Tumor and reference DNA were pooled, mixed with 50 µg Cot-1 DNA (Invitrogen) and concentrated to a volume of 20 µl in a vacuum centrifuge. DNA was then mixed with an equal amount of 2x formamide buffer, denaturated at 95°C for 5 min and preincubated at 37°C for 30 min in a waterbath.
Hybridization protocol
DNA samples were hybridized onto the array for 16-18 hours at 42°C utilizing the MAUI 4-bay hybridization system and MAUI Mixer A0 (BioMicro System, Salt Lake City, UT, USA). After hybridization arrays and MAUI Mixer were disassembled in 42°C 1xSSC and 0.05% SDS (wash solution 1) and subsequently washed two times in wash solution 1 for 5 minutes followed by two washes in 0.1xSSC (wash solution 2) for 5 minutes.
Scan protocol
Dried array slides were scanned using a ScanLite Express microarray scanner (Perkin Elmer, Wellesley, MA, USA). Raw image files of the arrays were processed using DeArray software (Chen et al, 1997) and resulting image data were imported into R environment using bioconductor packages (Gentleman RC et al., 2004) for further analysis.
