NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM1356018 Query DataSets for GSM1356018
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

Data table header descriptions
ID_REF
VALUE normalized log10 ratio Cy5/Cy3

Data table
ID_REF VALUE
1 -2.108377647e+000
2 0.000000000e+000
3 5.336905126e-002
4 8.091284571e-002
5 -8.228383190e-002
6 -5.631927267e-002
7 -1.946749490e+000
8 -5.010470464e-002
9 6.065680018e-002
10 1.034183704e-002
11 3.865370332e-002
12 -2.501598559e-002
13 -6.561934130e-002
14 -2.021137149e+000
15 2.022495300e-002
16 5.540416355e-002
17 4.160390265e-002
18 2.834995020e-002
19 1.757546967e-001
20 2.826794983e-002

Total number of rows: 42633

Table truncated, full table size 968 Kbytes.




Supplementary file Size Download File type/resource
GSM1356018_US22502540_251275013001_44k_T.txt.gz 10.9 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap