|
| Status |
Public on Mar 14, 2011 |
| Title |
CD133(+) cells of donor B vs. synthetic miRNA pool |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
CD133(+)/bone marrow
|
| Organism |
Homo sapiens |
| Characteristics |
sex: male
age: 73 years
tissue: bone marrow
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was isolated using a commercial kit (miRNeasy Mini kit, Qiagen) following the instructions of the manufacturer
|
| Label |
Hy5
|
| Label protocol |
1 µg total RNA was labelled using a commercial kit (miRCuryTM LNA microRNA Array Power labeling kit, Exiqon) following the instructions of the manufacturer
|
| |
|
| Channel 2 |
| Source name |
synthetic miRNA pool
|
| Organism |
synthetic construct |
| Characteristics |
reference: synthetic construct
|
| Extracted molecule |
other |
| Extraction protocol |
synthesized
|
| Label |
Hy3
|
| Label protocol |
2.5 fmol of each of 954 synthetic miRNAs were pooled and labelled using a commercial kit (miRCuryTM LNA microRNA Array Power labeling kit, Exiqon) following the instructions of the manufacturer total RNA was labelled using a commercial kit (miRCuryTM LNA microRNA Array Power labeling kit, Exiqon) following the instructions of the manufacturer
|
| |
|
| |
| Hybridization protocol |
1 µg of respective total RNA was mixed with 2.5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled by 3’ ligation. Total RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. Hybridization was performed as described by Bissels et al. (RNA 2009).
|
| Scan protocol |
Image capture was done with Agilent DNA-Microarray Scanner (Agilent, Santa Clara, CA)
|
| Description |
PMID
|
| Data processing |
Signal processing and quantification was done with ImaGene software version 8.0 (BioDiscovery, Los Angeles, CA). For each spot, the local signal was measured inside a fixed circle of 230-280 µm diameter, and background was measured outside the circle within specified rings 30 µm distant to the signal and 100 µm wide. Signal and background was taken to be the average of pixels between defined low and high percentages of maximum intensity with percentage parameter settings for low/high being 2/97% for signal and 0/97% for background. Local background was subtracted from the signal to obtain the net signal intensity and the mean of the net signal intensities of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was one-fold the mean background value in at least four of five donors in the CD133+ and/or CD34+CD133- cell population and where the miRNA was present in the UR. Subsequently,the array data was log2 transformed.
Only the miRNAs detected as present are contained in the data tables, and were used in subsequent data analysis including cluster analysis in TIGR MEV.
|
| |
|
| Submission date |
Jun 20, 2010 |
| Last update date |
Mar 14, 2011 |
| Contact name |
Ute Bissels |
| E-mail(s) |
ute.bissels@miltenyibiotec.de
|
| Organization name |
Miltenyi Biotec GmbH
|
| Department |
R&D
|
| Street address |
Friedrich-Ebert-Str. 68
|
| City |
Bergisch Gladbach |
| State/province |
NRW |
| ZIP/Postal code |
51429 |
| Country |
Germany |
| |
|
| Platform ID |
GPL8436 |
| Series (1) |
| GSE22460 |
Combined miRNA and mRNA analysis of CD133(+) and CD34(+) cells |
|
