naive B cell, isolated from peripheral blood of healthy donors by fluorescence-activated cell sorting
Treatment protocol
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. 2000 naïve B cells were isolated by magnetic cell separation, depleting CD27+ (memory B cells, T cell subpopulation) and CD11b+ (monocytes, macrophages, granulocytes, NK cells) cells, using the MACS system (Miltenyi Biotech, Bergisch Gladbach, Germany) followed by fluorescence-activated cell sorting (FACS) of IgD+ CD27- cells.
Extracted molecule
total RNA
Extraction protocol
The Purescript RNA Isolation Kit (Gentra) was applied using 80 μg glycogen (Roche) as a carrier and reducing all reagents to one-tenth of the amounts given in the standard protocol.
Label
biotin
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 μg 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 nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) in comparison with primary lymphoma cells of classical Hodgkin lymphoma cells and other B-non-Hodgkin lymphoma (B-NHL) samples and subsets of non-neoplastic B lymphocytes isolated from blood or tonsils.
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)
