|
| Status |
Public on Oct 12, 2012 |
| Title |
PG0028_GWP |
| Sample type |
genomic |
| |
|
| Source name |
Human B-cell chronic lymphocytic leukemia patient PG0028
|
| Organism |
Homo sapiens |
| Characteristics |
vh status: unmut
disease state: B-cell chronic lymphocytic leukemia
sample type: peripheral blood mononuclear cells
patient: PG0028
|
| Treatment protocol |
Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque (Seromed, Biochrom KG, Berlin, Germany) density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer (Becton Dickinson & Co, Sunnyvale, CA) with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome (Becton Dickinson). If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody (mAb) treatment (Becton Dickinson) followed by magnetic beads (Goat Anti-Mouse IgG Dynabeads, Dynal Biotech ASA, Oslo, Norway).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA extraction was performed using Wizard® Genomic DNA Purification kit according to the manufacturer's instructions (Promega).
|
| Label |
biotin
|
| Label protocol |
Biotinylated DNA were prepared according to the standard Affymetrix protocol starting from 250 nanograms of genomic DNA.
|
| |
|
| Hybridization protocol |
Following fragmentation, 90 micrograms of biotin-labeled DNA were hybridized for 16 hr at 49°C on GeneChip Human Mapping 250K NspI Arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
Human Mapping 250K NspI arrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix).
|
| Description |
legend: VH status = IgVH mutational status (Fais et al. J Clin Invest, 1998)
|
| Data processing |
Images were acquired using Affymetrix GeneChip® Operating System (GCOS 1.4). The raw data for individual SNPs were extracted from CEL files and converted into signal intensities using GTYPE 4.1 and Affymetrix Copy Number Analysis Tool (CNAT 4.0.1) softwares using the Hidden Markov Model algorithm with a genomic smoothing window set to 0. After the pre-processing, piecewise constant estimates of the underlying local DNA copy number (CN) variation was calculated using the DNAcopy Bioconductor package, which looks for optimal breakpoints using circular binary segmentation (CBS), (Olshen et al., Biostatistics 2004) and the median of the estimated pro?les was scaled back to a nominal multiplicity of two. After scaling, a k-means clustering algorithm was used on the cumulative profile of all the data to determine the thresholds for inferring discrete CN values. Inferred CN higher than 2.1 and 2.6 corresponded to gain and amplification whereas CN below 1.9 and 1.29 to loss and biallelic deletion, respectively.
|
| |
|
| Submission date |
Jun 08, 2012 |
| Last update date |
Oct 12, 2012 |
| Contact name |
Luca Agnelli |
| E-mail(s) |
[email protected], [email protected]
|
| Phone |
+390223903581
|
| Organization name |
IRCCS Istituto Nazionale dei Tumori
|
| Department |
Department of Advanced Diagnostics
|
| Street address |
Venezian 1
|
| City |
MILAN |
| ZIP/Postal code |
20133 |
| Country |
Italy |
| |
|
| Platform ID |
GPL3718 |
| Series (2) |
| GSE38613 |
Relevance of Chromosome 2p Gain in Early Binet Stage A Chronic Lymphocytic Leukemia (SNP) |
| GSE38618 |
Relevance of Chromosome 2p Gain in Early Binet Stage A Chronic Lymphocytic Leukemia |
|
