tissue: primary lymphoma cells laser-microdissected from a patient diagnosed with ALK-negative anaplastic large cell lymphoma (ALK- ALCL)
Treatment protocol
Five m-thick frozen sections of lymph nodes from lymphoma patients were mounted on membrane-covered slides (PALM, Bernried, Germany), incubated with hematoxylin containing 200 U/ml RNase inhibitor (Roche, Mannheim, Germany) for 4 minutes, washed in molecular biology grade water for 2 minutes, incubated in 2% eosin for 15 seconds, washed again, and dried at 37°C for 3 hours. Microdissection was performed using the Laser Microdissection and Pressure Catapulting (LMPC) technique with an UV-laser beam (PALM). Lymphoma cells were microdissected as single cells or groups of two to ten cells according to their characteristic morphological criteria concerning nuclear shape and diameter, chromatin distribution, nucleoli, and amount and density of cytoplasm. Cells were directly catapulted into Purescript lysis buffer (Gentra) and pooled in groups of 1000 – 2000 lymphoma 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)
