tissue: Neuroblastoma histology: stroma poor tumor stage: 4 patient nationality: Italian gender: male group: G3
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted overnight with lysis buffer containing K proteinase (0.2 mg/ml) followed by standard phenol/chloroform extraction. DNA content and quality were measured by Biophotometer (Eppendorf)
Label
cy5
Label protocol
We followed the "Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol”, v. 5.0 (Agilent Technologies). Briefly, 2 ug of sample and reference genomic DNAs (for reference were used Human Genomic DNA: male Cat G1471, female Cat G1521, Promega, Madison, WI) were separately digested with AluI/RsaI restriction enzyme mix (Promega). Fragmented DNA was labeled by direct enzymatic incorporation of fluorescent tags. Genomic DNA was labeled with Cy3-dUTP or Cy5-dUTP (Perkin Elmer) using the Random-Primed Bioprime DNA Labeling kit (Invitrogen Life Technologies). Briefly, 50 ul reaction mix containing dATP, dGTP and dTTP (120 ueach), dUTP (60 uM) and Cy3-dUTP (60 uM) or Cy5-dUTP (60 uM) was incubated with Klenow Fragment (40 units) at 37°C for 2 hrs. Unincorporated nucleotides were removed on Microcon YM-30 filters (Millipore) according to the manufacturer’s protocol. Dye incorporations of post-labeling products were measured by NanoDrop 1000 (ThermoScientific) and the parameters that predicted successful hybridization were a minimum Cy3 incorporation of 0.5 pmol/ul and Cy5 incorporation of 0.3 pmol/ul.
Genomic DNA was extracted overnight with lysis buffer containing K proteinase (0.2 mg/ml) followed by standard phenol/chloroform extraction. DNA content and quality were measured by Biophotometer (Eppendorf)
Label
cy3
Label protocol
We followed the "Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol”, v. 5.0 (Agilent Technologies). Briefly, 2 ug of sample and reference genomic DNAs (for reference were used Human Genomic DNA: male Cat G1471, female Cat G1521, Promega, Madison, WI) were separately digested with AluI/RsaI restriction enzyme mix (Promega). Fragmented DNA was labeled by direct enzymatic incorporation of fluorescent tags. Genomic DNA was labeled with Cy3-dUTP or Cy5-dUTP (Perkin Elmer) using the Random-Primed Bioprime DNA Labeling kit (Invitrogen Life Technologies). Briefly, 50 ul reaction mix containing dATP, dGTP and dTTP (120 ueach), dUTP (60 uM) and Cy3-dUTP (60 uM) or Cy5-dUTP (60 uM) was incubated with Klenow Fragment (40 units) at 37°C for 2 hrs. Unincorporated nucleotides were removed on Microcon YM-30 filters (Millipore) according to the manufacturer’s protocol. Dye incorporations of post-labeling products were measured by NanoDrop 1000 (ThermoScientific) and the parameters that predicted successful hybridization were a minimum Cy3 incorporation of 0.5 pmol/ul and Cy5 incorporation of 0.3 pmol/ul.
Hybridization protocol
Labelled DNAs were hybridized to whole-genome oligonucleotide 1x244K or 2x105K or 4x44K microarrays (Agilent Technologies) following the manufacture's instructions ("Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol”, v. 5.0 (Agilent Technologies). Briefly, fluorescent-labeled reference and tumor DNAs (3 ug each) were mixed with 50 ug of human Cot-1 DNA (Invitrogen Life Technologies) and control targets (Agilent Technologies). Slides were hybridized in SureHyb gasket (Agilent Technologies) placed in rotisserie (20 RPM rotation speed) in hybridization oven at 65°C for 40 hrs. After hybridization, the slides were washed with Oligo aCGH Wash Buffer 1(Agilent Technologies) at room temperature for 5 min, with Oligo aCGH Wash Buffer 2 (Agilent Technologies) at 37°C for 1 min, with the Stabilization solution (Agilent) for 1 minute at room temperature, then dried immediately by rinsing slides into Drying solution (Agilent) for 30 seconds.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using two color scan setting for 1x244k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel 1 is set to Red & Green, Red PMT is set to 100%, Green PMT is set to 100%) . Microarray performance was assessed by QC metric tool.
Description
aCGH analysis
Data processing
The scanned TIFF images were loaded into the Feature Extraction (v9.5 Agilent technologies), Feature Extraction files were analyzed by CGH Analytics Software (v. 3.5.14 Agilent Technologies) applying Z-Score algorithm.
