|
| Status |
Public on Nov 01, 2013 |
| Title |
MPI-932_post_GC_B_cells |
| Sample type |
RNA |
| |
|
| Source name |
post_GC_B_cells
|
| Organism |
Homo sapiens |
| Characteristics |
type: post_GC_B_cells
tumor_cell_line: NA
tumor: NA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Post GC memory B cells were isolated from peripheral blood samples of healthy individuals. For the isolation, FACS sorting employing antibodies against CD19 and CD27 was used. RNA was isolated using RNeasy Mini Kit (QIAGEN) and the concentration was determined photometrically (NanoDrop). The quality of the RNA was assessed based on the presence of the tht 18S and 28S tRNA by analysis of an aliquot in the Agilent BioAnalyzer (Agilent)
|
| Label |
biotin
|
| Label protocol |
For Affymetrix GeneChip hybridization, 5 microgram of total RNA was employed to generate a double-stranded cDNA with the help of the INVITROGEN Superscript kit. After removal of small molecular components (Affymetrix Clean-up Module), Biotin-labelled cRNA was synthesized by application of the ENZO Labeling kit (T7 RNA polymerase). The concentration of the produced cRNA was determined photometrically (NanoDrop) and the size range of the labelled products was evaluated by BioAnalyzer (Agilent) measurement before and after fragmentation
|
| |
|
| Hybridization protocol |
Affymetrix hybridization was carried on U133A GeneChips according to the manufacturer's recommendations. In brief, 15 microgram of Biotin-labelled and fragmented cRNA were mixed with 20x GeneChip Eukaryotic Hybridization Control (Affymetrix) and supplemented with B2 control oligonucletide, salmon sperm DNA, acetylated bovine serum albumin and hybridization buffer and water making up a total volume of 300 microliter
|
| Scan protocol |
After hybridization of night (16 hours, 45° C) the hybridization mixture was removed from the GeneChip and washing and staining of the GeneChips was performed in a fluidics station (Affymetrix). The detection of the Biotin-labeled cRNA was mediated by a streptavidin-phycoerythrin complex. The measurement of the fluorescence intensity was carried out in an Agilent scanner (GA2500)
|
| Data processing |
Probe level normalization was done using the calibration and variance stabilization method by Huber et al. (Bioinformatics 2002, PMID: 12169536). Probe-set summarization was performed using the median polish method on the normalised data (Irizarry et al. Nucleic Acids Research 2003, PMID: 12582260)
|
| |
|
| Submission date |
Jan 23, 2013 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Maciej Rosolowski |
| E-mail(s) |
maciej.rosolowski@imise.uni-leipzig.de
|
| Organization name |
Institute for Medical Informatics, Statistics and Epidemiology (IMISE)
|
| Street address |
Härtelstr. 16-18
|
| City |
Leipzig |
| ZIP/Postal code |
04107 |
| Country |
Germany |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE43677 |
Massive Transcriptional Perturbation in Subgroups of Diffuse Large B-cell Lymphomas |
|
| Relations |
| Reanalyzed by |
GSE86363 |
