|
| Status |
Public on Jan 01, 2014 |
| Title |
CLL_Immobilized_rep16 |
| Sample type |
RNA |
| |
|
| Source name |
CLL
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Peripheral blood
gender: male
cell type: B-CLL
treatment: co-stimulated with immobilized anti-IgM
|
| Treatment protocol |
In selected experiments, CLL cells co-stimulated with immobilized anti-IgM were cultured in the presence or not of R406 4mM (Axon Medchem, The Netherlands). CLL cells were also cultured in the presence or not of 7.5 μg/ml phosphorothioate CpG-ODN oligonucleotide 2006 (5′-TCG TCG TTT TGT CGT TTT GTC GTT-3′; CpG-ODN2006) (Life Technologies, CA) for 20 hours, as previously reported.27, 28
|
| Growth protocol |
Freshly isolated CLL cells cultured (1 × 107 cells/ml) in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine and 1 mM sodium pyruvate (Life Technologies, Carlsbad, CA, USA). CLL cells (n = 49/76) were also co-stimulated for 20h with 1 × 107/mL Dynabeads M-450 Epoxy (Life Technologies) coated with 10 μg goat anti-human IgM (immobilized anti-IgM) (SouthernBiotech, Birmingham, AL).27, 28 The coating procedure was done according to the manufacturer's instructions (Life Technologies).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from purified CLL cells and normal peripheral blood B cells of healthy donors using the TRIZOL Reagent (Life Technologies) and validated for integrity and purity using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Single-color hybridization microarray experiments for miRome were performed with 100 ng total RNA/sample labeled with Cyanine(Cy)-3 dye using the microRNA Complete Labeling System & Hyb Kit (Agilent Technologies).
|
| |
|
| Hybridization protocol |
Cy3-labeled RNA was hybridized to the Human microRNA microarray Version 3 from the Sanger database v12.0 (Agilent Technologies).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Normalized signal intensities provided in Series supplementary file miRNA_processed_data.txt.
|
| |
|
| Submission date |
Nov 26, 2013 |
| Last update date |
Jan 01, 2014 |
| Contact name |
daniela marconi |
| E-mail(s) |
[email protected]
|
| Organization name |
CRO AVIANO
|
| Street address |
Via franco Gallini 2
|
| City |
Aviano |
| ZIP/Postal code |
33081 |
| Country |
Italy |
| |
|
| Platform ID |
GPL11487 |
| Series (2) |
| GSE52775 |
Gene expression study in CLL of B-cell receptor triggering (miRNA study) |
| GSE52776 |
Gene expression study in CLL of B-cell receptor triggering |
|
