Pooled DNA from the blood of 10 normal male individuals
Biomaterial provider
VUMC, Amsterdam
Treatment protocol
none
Growth protocol
n.a.
Extracted molecule
genomic DNA
Extraction protocol
DNAzol (Invitrogen) according to manufacturer's protocol
Label
Cy5
Label protocol
DNA labeling protocol available via www.vumc.nl/microarrays Also described in Van den IJssel et al, Nucleic Acids Res. 2005 Dec 16;33(22):e192
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 100 µg Cot1 DNA used in hybridization mix
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 Yeast tRNA (100 µg/µl, Invitrogen) 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), 50% (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). Hybridisation 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 G2505B (Agilent Technologies).
Data processing
Spot analysis and quality control was fully automated using BlueFuse version 3.1 (BlueGnome, Cambridge, UK). Spots were excluded when the quality flag was less than 1 or the Confidence value less than 0.1. Oligonucleotides from the human library were mapped to the human genome build NCBI35. Oligonucleotide sequences and mapping have been made accessible by Compugen (San Jose, CA, USA) and The Sanger Institute (Hinxton, Cambridge, UK), respectively, via www.ensembl.org. A unique chromosomal position was identified for 26845 of 28830 oligonucleotides in the human library. Oligonucleotides were excluded when they mapped to more than one position in the genome or showed one or more mismatches with regard to the current build. Log2ratio’s of spots that were not excluded after quality flagging and mapping were normalized global to the median value of the chromosome 2 log2ratio's. Weighted moving average values were then calculated using a triangular function and a window of 250 kb as described (Barrett,M.T. et al. (2004) Proc.Natl.Acad.Sci.U.S.A, 101, 17765-17770.).
