Agilent cgh analytics version 6

Array-based CGH was performed using an Agilent Human Whole Genome CGH 8 × 60 K microarray (Agilent Technologies). Labeling and hybridization were performed using protocols provided by the manufacturer (Cancer Sci. 2013; 104; 631-638). Briefly, 0.5 μg of test or reference DNA was digested with Alu I and Rsa I (Promega, Madison, WI) and purified with the QIAprep Spin Miniprep kit (QIAGEN). Test and reference DNA samples were labeled by random priming with either Cy3-dUTP or Cy5-dUTP using the Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies). Following the labeling reaction, individually labeled test and reference samples were combined and concentrated using Amicon Ultra-0.5 centrifugal filters (Millipore, Billerica, MA). After denaturing the probe and preannealing it to human Cot-1 DNA, samples were hybridized at 65°C and 20 rpm rotation for 24 hours in a DNA Microarray Hybridization Oven (Agilent Technologies). Samples were washed in wash buffer 1 at room temperature for 5 minutes and wash buffer 2 at 37°C for 1 minute using Agilent Oligo CGH washes. All slides were scanned on an Agilent DNA microarray scanner. Data were obtained using Agilent Feature Extraction Software 9 and analyzed with Agilent CGH Analytics Version 6.5 software using the ADM-2 statistical algorithms with 6.0 sensitivity thresholds.