Sureprint g3 human cgh microarray 4 180k

Genomic DNA was extracted from FFPE tissue using a QIAamp, DNA micro kit (Qiagen, Hilden, Germany). Genomic DNA and human reference DNA (Promega) were labeled with cyanine 5 (Cy5) and cyanine 3 (Cy3), respectively, using the Genomic DNA High-Throughput ULS Labeling Kit (Agilent Technologies, Santa Clara, CA, USA) and co-hybridized onto a Sureprint G3 Human CGH microarray 4×180K (Agilent Technologies) following manufacturer’s recommendations. Data were analysed using Agilent Genomic Workbench software v7.0, or by Cytogenomics software (v2.9.2.4, Agilent), and expressed according to the human reference genome hg19 (GRCh37, Genome Reference Consortium Human Reference 37). The identification of aberrant copy number segments was based on ADM-2 segmentation algorithm with a threshold of 6.0.

aCGH was performed using genomic DNA isolated from primary tumors (Spanish cohort) and karyotypically normal reference genomic DNA (Promega, Madison, WI). The analysis was performed using Agilent Oligonucleotide Human Genome 180k CGH arrays. DNA was extracted from tumors using the QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA). The Genomic DNA ULS labeling kit for FFPE Samples (Agilent Technologies, Inc., Palo Alto, CA) was used to chemically label 500ng of DNA with either ULS-Cy5 (tumor) or ULS-Cy3 dye (normal/reference DNA) following the manufacturer’s protocol. Samples were hybridized to the Agilent SurePrint G3 Human CGH Microarray 4×180K. DNA samples were hybridized to the array using an Agilent microarray hybridization chamber for 40 hours in a Robbins Scientific oven with rotation at 20 rpm at 65°C. Post-hybridization, the slides were washed and scanned using an Agilent DNA microarray scanner. CGH Analytics software (version 3.4, Agilent Technologies, CA) was used to analyze the aCGH data.