Phenol/Chloroform Extraction Protocol: 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: 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, 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; Valencia, CA) and labeled 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). Cy3: 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).
gender: F sample type: Female human genomic DNA, Promega, G152A
Extracted molecule
genomic DNA
Extraction protocol
Phenol/Chloroform Extraction Protocol: 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
Cy3
Label protocol
Cy5: 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, 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; Valencia, CA) and labeled 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). Cy3: 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).
Hybridization protocol
According to Agilent user guide.
Scan protocol
Dried array slides were scanned using the DNA Microarray Scanner (Agilent, Santa Clara, CA).
Data processing
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.