Microcon ym 30 filter units

Array-CGH analysis was done by Agilent sure print G3 Hmn CGH 2x 400K arrays (Agilent Technologies, Santa Clara, USA) following manufacturer protocol. Briefly, 500ng patient’s DNA and also reference DNA of the same sex (Promega, Madison, USA) were digested with RsaI (Promega, Madison, USA) and AluI (Promega, Madison, USA) for 2hrs at 37ºC. Furthermore, these samples were labeled by random primers through Agilent labeling kit (Agilent Technologies, Santa Clara, USA) following manufacturer guidelines. Patient’s DNA was labeled with Cy5-dUTP whereas reference DNA was labelled with Cy3-dUTP. Labeled products were purified by Microcon YM-30 filter units (Millipore, Billerica, USA). Patient and reference DNAs were mixed with Cot-1 DNA (Invitrogen, USA) blocking agent and hybridization buffer following by Agilent instructions. After denaturation at 95ºC and pre-annealing at 37ºC, hybridization was done at 65ºC for 40hrs. After the two washing steps, array was analyzed through Agilent Scanner (G2505C) and Feature Extraction Software (V.1.5.1.0). Data was analyzed by using Partek Genomics Suite Software, Cytogenomics Software (V.2.0.6.0; Agilent Technologies, USA) and publicly available DGV.

The aCGH analysis was performed as per manufacturer's protocol
using Agilent SurePrint G3 Human CGH 2x400K arrays, Agilent
labeling kit (Agilent Technologies, USA). Briefly, 500ng of the
reference and sample DNA were digested at 37°C by RsaI and AluI
(Promega, USA) for 2h. Sample and reference DNA were labeled
with Cy3-dUTP (Green) and Cy5-dUTP (Red) respectively. Labeled
samples were purified and size selected using Microcon YM-30
filter units (Millipore, Billerica, Massachusetts, USA). Cot-1 DNA
(Invitrogen, Carlsbad, California, USA), hybridization buffer and
blocking agent were mixed with labeled DNA and denaturation
was performed at 95�C for 5min and maintained at 37°C, before
hybridization to the array at 65°C for 40 ± 2h at 20rpm. Microarray
slides and gaskets were disassembled and washed. Slides were
briefly rinsed with anhydrous acetonitrile followed by a final wash
with stabilizing and drying solution. Chip scanning, image analysis,
and data extraction were performed on an Agilent Scanner
(G2505C), and Agilent's Feature Extraction software (V.1.5.1.0)
respectively. Agilent CytoGenomics v2.7 software was used to
visualize, detect and analyze aberrations.