|
| Status |
Public on Aug 15, 2011 |
| Title |
fresh-frozen tissue_MDT_EP113 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
brain/ependymoma
|
| Organism |
Homo sapiens |
| Characteristics |
gender: M
age: 16
localization: posterior fossa
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Extraction of high molecular weight DNA from frozen tumor samples of DKFZ cohort was carried out as previously described (Pfister et al., 2009). Briefly, genomic DNA from peripheral blood mononuclear cells of healthy donors (pool of ten male and female donors, respectively, age 25-40 years) used as a control was isolated by use of the Qiagen DNA Blood Midi-Kit (Hilden, Germany).
|
| Label |
Cy3
|
| Label protocol |
Cell-line DNA and reference DNA from healthy donors were differentially labeled with Cy3-/ Cy5-conjugated dCTP by use of a BioPrime DNA Labeling Kit (Invitrogen, Karlsruhe, Germany).
|
| |
|
| Channel 2 |
| Source name |
reference DNA from healthy donors
|
| Organism |
Homo sapiens |
| Characteristics |
reference: DNA from healthy donors
tissue: peripheral blood
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Extraction of high molecular weight DNA from frozen tumor samples of DKFZ cohort was carried out as previously described (Pfister et al., 2009). Briefly, genomic DNA from peripheral blood mononuclear cells of healthy donors (pool of ten male and female donors, respectively, age 25-40 years) used as a control was isolated by use of the Qiagen DNA Blood Midi-Kit (Hilden, Germany).
|
| Label |
Cy5
|
| Label protocol |
Cell-line DNA and reference DNA from healthy donors were differentially labeled with Cy3-/ Cy5-conjugated dCTP by use of a BioPrime DNA Labeling Kit (Invitrogen, Karlsruhe, Germany).
|
| |
|
| |
| Hybridization protocol |
Hybridization was carried out in a GeneTAC Hybridization Station (Genomic Solutions, Oberhaching, Germany) for 36 hr at 378C.
|
| Scan protocol |
We scanned hybridized microarrays at a 5-mm resolution and variable PMT voltage to obtain maximal signal intensities with <0.1% probe saturation,a count ratio of 0.8–1.2 (Cy3/Cy5), and maximal congruence of histogram curves, using a Gene-Pix 4000B microarray scanner (Axon Instruments)
|
| Data processing |
Data were filtered according to signal/background ratio (>3.0), mean/median spot intensity (<0.3), and replicate standard deviation (<0.25) and normalized by print-tip loess. To identify regions of similar genomic status within the array-CGH data, we applied the segmentation software GLAD. Imbalances with log2 ratios of less than -1.0 were scored as putative homozygous deletions, because this threshold corresponds to an average copy number of less than 1, suggesting the presence of at least one subpopulation of cells with homozygous deletions. Gains with log2 ratios higher than 1.0 were scored as amplifications. Chromosomal mapping information was based on Ensembl (v49) and position of candidate genes was verified using Ensembl v52.
|
| |
|
| Submission date |
Feb 15, 2011 |
| Last update date |
Aug 15, 2011 |
| Contact name |
Hendrik Witt |
| E-mail(s) |
h.witt@dkfz.de
|
| Phone |
+496221424594
|
| Fax |
+496221424639
|
| Organization name |
German Cancer Research Center
|
| Department |
Molecular Genetics
|
| Lab |
Prof. Peter Lichter
|
| Street address |
INF 580
|
| City |
Heidelberg |
| State/province |
BW |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13171 |
| Series (2) |
| GSE27286 |
Human ependymoma samples, Subgrouping [aCGH - German Cancer Research Center human 33K BAC array] |
| GSE27287 |
Delineation of Two Clinically and Molecularly Distinct Subgroups of Posterior Fossa Ependymoma. |
|
