|
| Status |
Public on Oct 27, 2009 |
| Title |
liver vs. synthetic miRNA pool (2) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
liver
|
| Organism |
Mus musculus |
| Characteristics |
strain: CD1
gender: female
age: 8 weeks
tissue: liver
type: WT
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA as described in: Chomczynski P. (1993). A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques. 1993 Sep;15(3):532-4, 536-7"
|
| Label |
Hy5
|
| Label protocol |
5 µg total RNA was labelled using a commercial kit (miRCuryTM LNA microRNA Array labeling kit, Exiqon) following the instructions of the manufacturer
|
| |
|
| Channel 2 |
| Source name |
synthetic miRNA pool (miRXploreTM Universal Reference, Miltenyi Biotec)
|
| Organism |
synthetic construct |
| Characteristics |
synthetic miRNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
n/a
|
| Label |
Hy3
|
| Label protocol |
5 fmol of each of 816 synthetic miRNAs were pooled and labelld using a commercial kit (miRCuryTM LNA microRNA Array labeling kit, Exiqon) following the instructions of the manufacturer
|
| |
|
| |
| Hybridization protocol |
5 µg of respective total RNA was fluorescently labelled by 3’ ligation. Total RNA was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 5 fmol of each of 816 non redundant miRNAs sequences and miRControl 3 sequences. Hybridization was performed using an automated hybridization machine (a-Hyb, Miltenyi Biotec).
|
| 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).
|
| |
|
| Submission date |
Apr 24, 2009 |
| Last update date |
Jun 26, 2012 |
| 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 |
GPL8437 |
| Series (1) |
|
