|
| Status |
Public on Sep 05, 2012 |
| Title |
CD34neg_Donor_H |
| Sample type |
RNA |
| |
|
| Source name |
CD34neg, Donor H
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: CD34neg
gender: female
mobilization: Cy
|
| Treatment protocol |
MS patients (4 donors) and age matched healthy donors (5) underwent stem cell mobilization by G-CSF (2x5μg/kg/day). An additional set of MS patients (4 donors) receiced G-CSF (5μg/kg/day) plus Cyclophosphamide (Cy, 4g/m2).
|
| Growth protocol |
ex vivo isolated cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
White blood cells, containing the CD34+ cell fraction, were collected by leucocytapheresis from peripheral blood, frozen and stored in liquid nitrogen. All samples were thawed and processed at one center and CD34+ cells purified by magnetic bead separation using the autoMACS system (Miltenyi). Purity and viability of CD34+ cells was analyzed by Fluorescence Activated Cell Sorter (FACS). Total cellular RNA and DNA were extracted with TRIzol reagent. RNA samples were checked for quantity, purity and integrity of the 18S and 28S ribosomal bands by capillary electrophoresis using the Agilent 2100 bioanalyzer (nanoassay).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 700 ng RNA using the Low RNA Input linear Amplification Kit Plus, One Color protocol (Agilent Techologies, Inc. 2007; Cat. N°: 51885339) and the Agilent RNA SpikeIn Kit for One color (Agilent Techologies, Inc. 2007; Cat. N°: 51885282). Quantity and efficiency of the labeled amplified cRNA were determined using the NanoDrop ND1000 UVVIS Spectrophotometer version 3.2.1.
|
| |
|
| Hybridization protocol |
Cy3-labelled cRNA was fragmented following manufacturers instructions and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 10 rpm and 65°C in the Hybridization Oven (Agilent). Washing and staining of the arrays were done according to the manufacturers recommendation.
|
| Scan protocol |
Cy3 intensities were detected by onecolor scanning using an Agilent DNA microarray scanner (Agilent) at 5 micron resolution. Scanned image files were visually inspected for artifacts and then analyzed
|
| Description |
Gene expression hematopoietic progenitor cells
|
| Data processing |
Slides were scanned using Agilent Microarray Scanner (G2565BA) followed by data extraction by Agilent Feature Extraction software version 9.5.3. with default settings for oligonucleotide expression arrays. Intensity data were processed with R and Bioconductor packages to obtain quantile-normalized and log2-transformed intensity values. Calculated p-values for differential expression based on a moderated t-statistic were corrected for multiple testing using the Benjamini-Hochberg method. Samples were grouped according to the mobilization regimen followed (G‑CSF vs G‑CSF/CY). Comparisons were made for MS affected patients (G-CSF) vs healthy donors and G-CSF/CY vs G-CSF mobilization for MS patients only, respectively.
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| |
|
| Submission date |
Mar 04, 2011 |
| Last update date |
Sep 05, 2012 |
| Contact name |
Christian Schulze |
| E-mail(s) |
christian.schulze@zmnh.uni-hamburg.de
|
| Organization name |
ZMNH
|
| Street address |
Falkenried 94
|
| City |
Hamburg |
| ZIP/Postal code |
20251 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6480 |
| Series (2) |
| GSE27688 |
Gene expression and epigenetic profiling of CD34+ hematopoietic progenitor cells in multiple sclerosis patients (expression) |
| GSE27694 |
Gene expression and epigenetic profiling of CD34+ hematopoietic progenitor cells in multiple sclerosis patients |
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