|
| Status |
Public on Jun 01, 2014 |
| Title |
NB33 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Tumor DNA
|
| Organism |
Homo sapiens |
| Characteristics |
Sex: male
age at diagnosis (days): 546
tumor stage: 1
tissue: neuroblastoma
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated using the DNA-Isolation Kit (Gentra) according to the manufacturer's protocol. A pool of normal male or female DNA (Promega) was used as reference.
|
| 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.
|
| |
|
| Channel 2 |
| Source name |
Promega Reference DNA
|
| Organism |
Homo sapiens |
| Characteristics |
Sex: M
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated using the DNA-Isolation Kit (Gentra) according to the manufacturer's protocol. A pool of normal male or female DNA (Promega) was used as reference.
|
| 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.
|
| |
|
| |
| Hybridization protocol |
Labelled DNA were hybridized to oligonucleotide 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 DNA (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 |
Arrays were scanned by using an Agilent G2567AA Scanner follwowing the manufacturer's protocol (Agilent Technologies).
|
| Description |
Agilent-012750 Human Genome CGH Microarray 44A (G4410A)
|
| 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). The circular binary segmentation algorithm was applied. Ref: PMID: 17234643
|
| |
|
| Submission date |
Mar 21, 2014 |
| Last update date |
Jun 01, 2014 |
| Contact name |
Jessica Theissen |
| E-mail(s) |
jessica.theissen@uk-koeln.de
|
| Phone |
+49 221 478 6843
|
| Organization name |
University of Cologne
|
| Department |
Department of Pediatric Oncology and Hematology
|
| Street address |
Kerpener Straße 62
|
| City |
Cologne |
| State/province |
NRW |
| ZIP/Postal code |
50924 |
| Country |
Germany |
| |
|
| Platform ID |
GPL2873 |
| Series (1) |
| GSE56109 |
Chromosome 17/17q gain and unaltered profiles in high resolution array-CGH are prognostically informative in neuroblastoma |
|
