|
| Status |
Public on Apr 03, 2013 |
| Title |
NL6 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
NL_CD138+ selected cells
|
| Organism |
Homo sapiens |
| Characteristics |
subject condition: normal donor
cell type: CD138+ selected cells from bone marrow aspirates
restriction enzyme used: MspI
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Following Ficoll-Hypaque gradient centrifugation, plasma cells obtained from the bone marrow were isolated from the mononuclear cell fraction by immunomagnetic bead selection using a monoclonal mouse anti-human CD138 antibody (Miltenyi-Biotec, Auburn, CA). Total More than 90 percent of the cells used for gene expression profiling were plasma cells, as shown by two-color flow cytometry using CD138+/CD45- and CD38+/CD45- markers.Genomic DNA was isolated using Puregene Cell and Tissue Kit according to the manufacturer instructions (Qiagene). Representation of the genome generated by digestion with either MspI or HpaII and amplification by ligation-mediated PCR according to the HELP protocol Khulan B et al. Genome Res. 2006 Aug;16(8):1046-55).
|
| Label |
Cy3
|
| Label protocol |
Random 9-mers pre-labeled with either Cy5 or Cy3
|
| |
|
| Channel 2 |
| Source name |
NL_CD138+ selected cells
|
| Organism |
Homo sapiens |
| Characteristics |
subject condition: normal donor
cell type: CD138+ selected cells from bone marrow aspirates
gdna digeted with: HpaII
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Following Ficoll-Hypaque gradient centrifugation, plasma cells obtained from the bone marrow were isolated from the mononuclear cell fraction by immunomagnetic bead selection using a monoclonal mouse anti-human CD138 antibody (Miltenyi-Biotec, Auburn, CA). Total More than 90 percent of the cells used for gene expression profiling were plasma cells, as shown by two-color flow cytometry using CD138+/CD45- and CD38+/CD45- markers.Genomic DNA was isolated using Puregene Cell and Tissue Kit according to the manufacturer instructions (Qiagene). Representation of the genome generated by digestion with either MspI or HpaII and amplification by ligation-mediated PCR according to the HELP protocol Khulan B et al. Genome Res. 2006 Aug;16(8):1046-55).
|
| Label |
Cy5
|
| Label protocol |
Random 9-mers pre-labeled with either Cy5 or Cy3
|
| |
|
| |
| Hybridization protocol |
See Roche NimbleGen website and Selzer RR, Richmond TA, Pofahl NJ,Green RD, Eis PS, et al. (2005) Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH. Genes Chromosomes Cancer 44: 305-319. for details.
|
| Scan protocol |
Scanning was performed using a GenePix 4000B scanner (Axon Instruments) as previously described in Selzer RR, Richmond TA, Pofahl NJ, Green RD, Eis PS, et al. (2005) Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH. Genes Chromosomes Cancer 44: 305-319
|
| Description |
CD138F
Methylation data from HELP assay
|
| Data processing |
Signal intensities at each HpaII amplifiable fragment were calculated as a robust (25% trimmed) mean of their component probe-level signal intensities. Any fragments found within the level of background MspI signal intensity, measured as 2.5 mean-absolute-differences (MAD) above the median of random probe signals, were categorized as “failed.” These “failed” loci therefore represent the population of fragments that did not amplify by PCR, whatever the biological (e.g. genomic deletions and other sequence errors) or experimental cause. On the other hand, “Methylated” loci were so designated when the level of HpaII signal intensity was similarly indistinguishable from background.PCR-amplifying fragments (those not flagged as either “methylated” or “failed”) were normalized using an intra-array quantile approach wherein HpaII/MspI ratios are aligned across density-dependent sliding windows of fragment size-sorted data (described in detail in Thompson et al, Bioinformatics 2008;24:1161-1167.)
|
| |
|
| Submission date |
Jan 29, 2013 |
| Last update date |
Apr 03, 2013 |
| Contact name |
Christoph Heuck |
| Organization name |
Albert Einstein College of Medicine
|
| Department |
Cancer Center
|
| Lab |
Verma Lab
|
| Street address |
1300 Morris Park Ave
|
| City |
Bronx |
| ZIP/Postal code |
10461 |
| Country |
USA |
| |
|
| Platform ID |
GPL6604 |
| Series (1) |
| GSE43860 |
DNA methylation in normal PC, MGUS, SMM and MM |
|
