sample origin: Primary mediastinal B-cell lymphoma case 2 cell type: primary lymphoma cells isolated by: laser-microdissection from a patient's biopsy involved by primary mediastinal B-cell lymphoma
Growth protocol
PRIMARY TUMOR SAMPLES: Five μm-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). Areas with at least 95% lymphoma cells were selected, so that these lymphoma cells could be microdissected in areas. Cells were directly catapulted into Purescript lysis buffer (Gentra) and pooled in groups of 1000 – 2000 lymphoma cells. CELL LINE SAMPLES: In vitro growing cell cultures were cooled at 4°C and 500-1000 living cells were immediately subjected to fluorescence-activated cell sorting directly into Purescript lysis buffer (Gentra).
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
1000-2000 microdissected tumor cells
Data processing
Most bioinformatics analyses were performed using GeneSpring 7.3.1 software (Agilent Technologies) with GC-RMA preprocessing of the raw expression data (CEL files). Further statistical analysis was done with the computing environment R. Additional software packages (affy, geneplotter) were taken from the Bioconductor project.
