DNA was extracted from formalin fixed, paraffin embedded tumor tissue according to protocols from the UCSF Waldman Laboratory, San Francisco, CA, USA (http://cc.ucsf.edu/people/waldman/Protocols/paraffin.html), with an additional purification step using Phase Lock Gel tubes (Eppendorf AG, Hamburg, Germany).
Label
Cy3
Label protocol
2-8 μg tumor DNA and 2 µg reference DNA (Promega Corporation, Madison, WI, USA) were labeled with Cy3-dCTP and Cy5-dCTP, using BioPrime Array CGH Genomic Labeling System (Invitrogen Life Technologies, Carlsbad, CA, USA).
DNA was extracted from formalin fixed, paraffin embedded tumor tissue according to protocols from the UCSF Waldman Laboratory, San Francisco, CA, USA (http://cc.ucsf.edu/people/waldman/Protocols/paraffin.html), with an additional purification step using Phase Lock Gel tubes (Eppendorf AG, Hamburg, Germany).
Label
Cy5
Label protocol
2-8 μg tumor DNA and 2 µg reference DNA (Promega Corporation, Madison, WI, USA) were labeled with Cy3-dCTP and Cy5-dCTP, using BioPrime Array CGH Genomic Labeling System (Invitrogen Life Technologies, Carlsbad, CA, USA).
Hybridization protocol
Hybridizations were conducted using the MAUI® Hybridization System (BioMicro systems Inc, Salt Lake City, UT, USA).
Scan protocol
The slides were scanned in an Agilent Microarray Scanner (Agilent Technologies).
Description
90_cy3
Data processing
TIFF images were analysed using the GenePixTM Pro. 4.1 software (Axon Instruments Inc., Foster City, CA, USA) and quantified data matrices were uploaded in the web based BioArray Software Environment software (BASE) (Saal et al., 2002). Positive and nonsaturated spots were background corrected using the median background intensities for each channel and log2 ratios were calculated from the background corrected intensities. Data were filtered for flagged features and spots below signal to noise ratio 3 for both channels were eliminated. Data was normalized using an implementation of a population based LOWESS algorithm (Staaf et al., 2007).