|
| Status |
Public on Nov 16, 2009 |
| Title |
NKT_B2 |
| Sample type |
RNA |
| |
|
| Source name |
NKT cells donor 2
|
| Organism |
Homo sapiens |
| Characteristics |
cells: NKT cells, isolated from peripheral blood by fluorescence-activated cell sorting
|
| Treatment protocol |
NKT cells were isolated from peripheral blood of five healthy adult donors. blood samples were immediately cooled at 0-4°C and cells were further processed and immediately sorted after staining at this low temperature to avoid changes in gene expression, due to RNA turnover or signalling effects of the antibodies used for staining. NKT cells from freshly isolated, heparinised blood were also isolated by MACS enrichment. Cells were stained with anti-V24JQ (clone 6B11)-PE and subsequently enriched with PE-Mircobeads (PE MicroBeads, Miltenyi Biotech, Bergisch Gladbach, Germany) followed by FACS of CD3+ V24JQ+ V11+ cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The Purescript RNA Isolation Kit (Gentra) was applied using 80 g glycogen (Roche) as a carrier and reducing all reagents to one-tenth of the amounts given in the standard protocol.
|
| Label |
SAPE (streptavidin-phycoerythrin)
|
| Label protocol |
The T7 RNA polymerase-based RiboAmpTM RNA Amplification Kit, version C (Arcturus, Mountain View, CA, USA) was used to amplify the RNA by in vitro transcription. In the first cDNA synthesis, 1.5 g T4 gene 32 protein (Ambion, Austin, USA) were added to the reaction to improve specificity and yield of the reaction. The first cDNA synthesis was followed by an in vitro transcription (IVT) and a second cDNA synthesis using random hexamers. 100 ng of the second cDNA synthesis product were used in the labeling reaction with the BioArray High Yield Transcription Labeling Kit (ENZO Life Science, Farmingdale, NY, USA) with the following modifications: the incubation-time was prolonged to 8 hours and the ENZO T7 RNA polymerase (150 U) was substituted by Stratagene T7 RNA polymerase (300 U) (Stratagene, La Jolla, CA, USA).
|
| |
|
| Hybridization protocol |
Fragmentation of cRNA, microarray hybridisation to Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays, washing steps of the microarrays was performed according to the Affymetrix protocol.
|
| Scan protocol |
Scanning was performed according to the Affymetrix protocol.
|
| Description |
Analysis of differential gene expression in primary human lymphoma cells of ALK-positive, ALK-negative and cutaneous anaplastic large cell lymphoma (ALCL) in comparison with primary lymphoma cells of classical Hodgkin lymphoma cells and subsets of non-neoplastic T and NK lymphocytes isolated from tonsils or blood.
|
| Data processing |
Probe level normalization was conducted using the variance stabilization method of Huber et al. (Huber et al., 2002, Bioinformatics 18 Suppl 1:S96-104)
|
| |
|
| Submission date |
Feb 16, 2009 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Ralf Küppers |
| E-mail(s) |
ralf.kueppers@uk-essen.de
|
| Phone |
0049 201 723 3384
|
| Fax |
0049 201 723 3386
|
| Organization name |
University of Duisburg-Essen, Medical School
|
| Department |
Institute for Cell Biology (Tumor Research)
|
| Street address |
Virchowstr. 173
|
| City |
Essen |
| ZIP/Postal code |
45122 |
| Country |
Germany |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE14879 |
Gene expression study of anaplastic large cell lymphomas: cellular origin, pathogenesis and relation to Hodgkin lymphoma |
|
| Relations |
| Reanalyzed by |
GSE65823 |
| Reanalyzed by |
GSE119087 |
