tumor samples used were frozen prior to extraction
Extracted molecule
genomic DNA
Extraction protocol
The version 2.0 protocol (August 2005) for Oligonucleotide Array-Based CGH for Genomic DNA Analysis was used. Three μg of Human Genomic DNA from multiple anonymous male donors (Promega Corporation, Madison, USA) and 3 μg of tumor genomic DNA samples were digested with AluI (50 units) and RsaI (50 units) (Promega) for a minimum of 2 hours at 37C. Digestion quality was assessed by the DNA 1000 LabChip Kit (Agilent 2100 Bioanalyzer, Agilent Technologies), and the products purified using the Qiaquick PCR Cleanup Kit (Qiagen Inc., Cologne, Germany).
Label
cy3 and cy5 (dye-flip)
Label protocol
Labelling reactions were performed using the BioPrime Array CGH Genomic Labelling System (Invitrogen, CA, USA) according to the manufacturer’s directions. Briefly, the reference and sample DNA were labelled with 3 μl of 1.0 mM Cy5-dUTP or Cy3-dUTP (Perkin-Elmer Life and Analytical Sciences, Woodbridge, Canada), in a 10 x dUTP nucleotide mix of 1.2 mM each of dATP, dGTP, dCTP and 0.6 mM dTTP (Invitrogen) and purified using a Centricon YM-30 filter (Millipore, Billerica, USA).
Human Genomic DNA from multiple anonymous male donors. Purchased from Promega Corporation, Madison, USA. Cat# G1471.
Treatment protocol
no treatment was performed
Growth protocol
N/A
Extracted molecule
genomic DNA
Extraction protocol
The version 2.0 protocol (August 2005) for Oligonucleotide Array-Based CGH for Genomic DNA Analysis was used. Three μg of Human Genomic DNA from multiple anonymous male donors (Promega Corporation, Madison, USA) and 3 μg of tumor genomic DNA samples were digested with AluI (50 units) and RsaI (50 units) (Promega) for a minimum of 2 hours at 37C. Digestion quality was assessed by the DNA 1000 LabChip Kit (Agilent 2100 Bioanalyzer, Agilent Technologies), and the products purified using the Qiaquick PCR Cleanup Kit (Qiagen Inc., Cologne, Germany).
Label
cy3 and cy5 (dye-flip)
Label protocol
Labelling reactions were performed using the BioPrime Array CGH Genomic Labelling System (Invitrogen, CA, USA) according to the manufacturer’s directions. Briefly, the reference and sample DNA were labelled with 3 μl of 1.0 mM Cy5-dUTP or Cy3-dUTP (Perkin-Elmer Life and Analytical Sciences, Woodbridge, Canada), in a 10 x dUTP nucleotide mix of 1.2 mM each of dATP, dGTP, dCTP and 0.6 mM dTTP (Invitrogen) and purified using a Centricon YM-30 filter (Millipore, Billerica, USA).
Hybridization protocol
Probe mixture of Cy3 labelled sample DNA, Cy5 labelled reference DNA, 50 μl of 1.0mg/ ml of human Cot-1 DNA (Invitrogen, USA), 50 μl of Agilent 10X Blocking Agent and 250 μl of Agilent 2X Hybridization Buffer (Agilent Technologies, Inc.) was denatured at 100C for 1 minute 30 s and incubated at 37C for 30 minutes. The probe was applied to the array using an Agilent microarray hybridization chamber, and hybridized for 40 hours at 65C in a rotating oven (Robbins Scientific, Sunnyvale, USA) at 20 rpm. Dye swaps were carried out routinely.
Scan protocol
Arrays were washed according to the manufacturer’s recommendations, air dried, and scanned using an Agilent 2565AA DNA microarray scanner (Agilent Technologies, Inc).
Description
The identification of tumor-specific genomic profiles using array CGH.
Data processing
Normalization strategy involved averaging the dye-flip Cy5/Cy3 and Cy3/Cy5 ratios for a sample; algorithm used was the ADM-1 proivded by Agilent analytics 3.4 with a threshold of 10.0. Raw data and normalized data files for the individual dye-swap hybridizations are linked as supplementary files at the foot of the Sample record.
