normal DNA pooled from the blood of ten healthy males
Extracted molecule
genomic DNA
Extraction protocol
DNAzol (Invitrogen) according to manufacturer's protocol
Label
Cy5
Label protocol
According to snijders et al., 2001
Hybridization protocol
Hybridization protocol available via www.vumc.nl/microarrays Also described in Van den IJssel et al, Nucleic Acids Res. 2005 Dec 16;33(22):e192 no prehyb solution used
For preparation of the hybridization mixture 50 µl of Cy3-labeled test DNA, 50 µl of Cy5-labeled reference DNA and 100 µg Human Cot-1 DNA (Invitrogen) were mixed and precipitated using 0.1 volume of 3 M NaAc pH 5.2 and 2.5 volume of ice-cold absolute ethanol. After mixing by inversion the DNA was collected by centrifugation for 30 min at 20,000 g and 4°C, the supernatant aspirated and the pellet air-dried for approximately 5-10 min. The pellet was then dissolved in 13 µl water and 26 µl 20% SDS taking care to prevent foam formation. After incubating at room temperature for 15 min 91 µl of Master mix (14.3 % (w/v) dextran sulphate (USB), 71% (v/v) formamide (Invitrogen), 2.9 X SSC pH 7.0 (Sigma)) was added and gently mixed. The hybridization solution was then incubated at 73°C for 10 min to denature the DNA and subsequently at 37°C for 60 min to allow the Cot-1 DNA to block repetitive sequences. Hybridization and washing was done automatically using a GeneTAC/HybArray12 hybstation (Genomic Solutions / Perkin Elmer). Hybridization was for 38 h at 37°C. Subsequently slides were washed 6 cycles (flow for 10 s, hold for 20 s) with 50% (v/v) formamide, 2X SSC, 2 cycles with phosphate-buffer (0.1 M Na2HPO4/NaH2PO4, pH 8.0, 0.1% (v/v) Igepal CA630 (Sigma)), 2 cycles with 0.2 X SSC (Sigma) and 2 cycles with 0.1 X SSC. Slides were then taken out of the hybstation and briefly rinsed in 0.01 X SSC, dried by centrifugation for 3 min at 1000 g and scanned using a Microarray Scanner G2565AB (Agilent Technologies).
Data processing
Spot analysis and quality control was fully automated using BlueFuse version 3.4 (BlueGnome, Cambridge, UK). Spots were excluded when the quality flag was less than 1, the Confidence value less than 0.3 when stdev was larger than 0.2 or when no chromosomal mapping information was available. Log2ratio’s of spots that were not excluded after quality flagging and mapping were normalized to their median value.
