cells: resting cytotoxic T-cells, isolated from tonsils by fluorescence-activated cell sorting
Treatment protocol
Tonsils for the isolation of resting cytotoxic T-cells (CD8R) were obtained from patients undergoing routine tonsillectomy. Tonsils 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. CD8+ T-cells were isolated by MACS depletion of CD4+, CD14+, CD16+, CD19+, CD36+, CD56+, CD123+, TCR γ/δ, and CD235a+ (CD4+ T cells, γ/δ T cells, B cells, NK cells, dendritic cells, monocytes, granulocytes, and erythroid cells)(CD8+ T cell isolation kit II, Miltenyi Biotech, Bergisch Gladbach, Germany) followed by FACS-sorting of 2000 CD3+ CD8+ HLA-DR- cells 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)
