Promega Genomic DNA Purification Other: Genomic DNA obtained from multiple anonymous donors was purified by the method described in Sambrook et al. Greater than 90% of the DNA is longer than 50kb in size as measured by pulsed-field gel electrophoresis. (See: http://www.promega.com/)
Label
cy3
Label protocol
Cy3 Labeling Protocol Other: Five micrograms of reference DNA of the opposite gender (human genomic DNA, Promega, San Luis Obispo, CA) were digested with the restriction enzymes AluI and RsaI (10U/uL; Invitrogen, Carlsbad, CA). Digested DNA was purified using QIAprep Spin Miniprep Kit (QIAGEN, Valencia, CA) and labeled using the BioPrime Array CGH Genomic Labeling kit (Invitrogen, Carlsbad, CA) according to the manufacturer'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 Cy3-dUTP (GE Amersham, Piscataway, NJ). Reference DNA was pooled, purified and concentrated to 79 µl using Vivaspin 500 concentrator (VS0122, 30k MWCO, Vivascience, Littleton, MA).
Phenol/Chloroform Extraction Protocol Other: Genomic tumor cell DNA was isolated from microdissected fresh frozen clinical tumor samples using proteinaseK digestion (P2308, Sigma-Aldrich, St. Louis, MO) and subsequent Phenol/Chlororform extraction.
Label
cy5
Label protocol
Cy5 Labeling Protocol Other: Five micrograms of genomic DNA were digested with the restriction enzymes AluI and RsaI (10U/uL; Invitrogen, Carlsbad, CA). Digested DNA was purified using QIAprep Spin Miniprep Kit (QIAGEN, Valencia, CA) and labeled using the BioPrime Array CGH Genomic Labeling kit (Invitrogen, Carlsbad, CA) according to the manufacturer'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 (GE Amersham, Piscataway, NJ). Tumor DNA was pooled, purified and concentrated to 79 µl using Vivaspin 500 concentrator (VS0122, 30k MWCO, Vivascience, Littleton, MA).
Hybridization protocol
Sample Hybridization Protocol Other: 25 µg Cot-1 DNA (Invitrogen, Carlsbad, CA), 1/10 volume of 10x Blocking Agent (Agilent, Santa Clara, CA) and an equal amount of 2× Hybridization Buffer (Agilent, Santa Clara, CA) were added to the labeled genomic DNA, denatured at 95°C for 5 min and preincubated at 37°C for 30 min in a water bath. Hybridizations were carried out on Human Genome CGH 105A Oligo Microarray glass slides (G4412A, Agilent, Santa Clara, CA). DNA samples were hybridized onto the array for 36~V40 hours at 65°C utilizing the DNA Microarray Hybridization Chamber SureHyb and Hybridization Oven (Agilent, Santa Clara, CA). After hybridization arrays were disassembled at room temperature, they were subsequently washed in wash solution 1 (0.5×SSPE and 0.005% N-Lauroylsarcosine) at room temperature for 5 min and wash solution 2 (0.1×SSPE and 0.005% N-Lauroylsarcosine) at 37°C for 1 min.
Scan protocol
Agilent Scanning Protocol Other: Dried array slides were scanned using the DNA Microarray Scanner (Agilent, Santa Clara, CA).
Description
No additional information.
Data processing
Agilent Data Processing Protocol Calculation Method: Raw image files of the arrays were processed using Feature Extraction software 8.1 using default CGH parameters (Agilent, Santa Clara, CA). Spot values were normalized using the default linear-lowess normalization.
