DNA was extracted from each breast cancer tissue sample using the PureGene kit (Gentra Systems Inc., Minneapolis, MN, USA).
Label
Cy3
Label protocol
The labeling and hybridization protocols described by Pinkel et al. were used with some modification to the labeling procedure. Briefly, 21 μl solution containing 500 ng normal DNA (reference DNA) or tumor DNA (test DNA), 20 μl BioPrime DNA Labeling System random primers solution (Invitrogen, Carlsbad, CA, USA), and water were combined and incubated for 5 min at 95 °C, and subsequently cooled on ice. After the addition of 5 μl 10x dNTPs labeling mix (1 mM dCTP, 2 mM dATP, 2 mM dGTP, 2 mM dTTP), 3 μl 1 mM Cy-3 or Cy-5 dCTP (GeneChem Inc., Daejeon, Korea), and 40 U BioPrime DNA Labeling System Klenow fragment (Invitrogen), the mixture was gently mixed and incubated overnight at 37 °C. The addition of 5 μl BioPrime DNA Labeling System Stop Buffer (Invitrogen) ended the reaction. After labeling, unincorporated fluorescent nucleotides were removed using QIAquick Spin columns (Qiagen, Germany). In one tube, Cy3-labeled sample and Cy5-labeled reference DNAs were mixed together, and 50 μg human Cot I DNA (Invitrogen), 20 μl 3M sodium acetate, and 600 μl cold 100% ethanol were added for DNA precipitation.
Channel 2 DNA was purchased from Promega Corporation: Human Genomic DNA, Female (#G1521)
Label
Cy5
Label protocol
The labeling and hybridization protocols described by Pinkel et al. were used with some modification to the labeling procedure. Briefly, 21 μl solution containing 500 ng normal DNA (reference DNA) or tumor DNA (test DNA), 20 μl BioPrime DNA Labeling System random primers solution (Invitrogen, Carlsbad, CA, USA), and water were combined and incubated for 5 min at 95 °C, and subsequently cooled on ice. After the addition of 5 μl 10x dNTPs labeling mix (1 mM dCTP, 2 mM dATP, 2 mM dGTP, 2 mM dTTP), 3 μl 1 mM Cy-3 or Cy-5 dCTP (GeneChem Inc., Daejeon, Korea), and 40 U BioPrime DNA Labeling System Klenow fragment (Invitrogen), the mixture was gently mixed and incubated overnight at 37 °C. The addition of 5 μl BioPrime DNA Labeling System Stop Buffer (Invitrogen) ended the reaction. After labeling, unincorporated fluorescent nucleotides were removed using QIAquick Spin columns (Qiagen, Germany). In one tube, Cy3-labeled sample and Cy5-labeled reference DNAs were mixed together, and 50 μg human Cot I DNA (Invitrogen), 20 μl 3M sodium acetate, and 600 μl cold 100% ethanol were added for DNA precipitation.
Hybridization protocol
The pellet was resuspended in 40 μl hybridization solution containing 50% formamide, 10% dextran sulfate, 2x SSC, 4% SDS, and 200 μg yeast tRNA. The hybridization solution was denatured for 10 min at 72 °C and was subsequently incubated for 1 hour at 37 °C to allow blocking of repetitive sequences. Hybridization was performed in slide chambers for 48 hours at 37 °C. After post-hybridization washes, arrays were rinsed, spin-dried.
Scan protocol
Arrays were scanned into two 16-bit TIFF image files using a GenePix 4200A two-color fluorescent scanner (Axon Instruments), and individual spots were analyzed with GenePix Pro 3.0 imaging software (Axon Instruments).
Description
SNU_Breast_CGH_S14
Data processing
The log2-transformed fluorescent ratios were calculated from background-subtracted median intensity values, and these ratios were used to perform normalization according to intensity normalization methods. We applied LOWESS normalization, a smoothing adjustment that removes intensity-dependent variation in dye bias.
