|
| Status |
Public on Oct 27, 2009 |
| Title |
liver and spike ins vs. synthetic miRNA pool (2) |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
liver + 75 synthetic RNAs (spike ins)
|
| Organism |
Mus musculus |
| Characteristics |
tissue: liver
|
| 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 |
0.5 µg total RNA and the spike ins (0.3125-20 fmol) were 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 (miRXploreTM Universal Reference, Miltenyi Biotec)
|
| Organism |
synthetic construct |
| Characteristics |
composition: 2.5 fmol of each of 889 synthetic miRNAs were pooled and labelled.
|
| Extracted molecule |
other |
| Extraction protocol |
n/a
|
| Label |
Hy3
|
| Label protocol |
2.5 fmol of each of 889 synthetic miRNAs were pooled and labelld using a commercial kit (miRCuryTM LNA microRNA Array Power labeling kit, Exiqon) following the instructions of the manufacturer
|
| |
|
| |
| Hybridization protocol |
0.5 µg of respective total RNA were mixed with 75 spike in synthetic RNAs (which are not expressed in mouse liver) and 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. The synthetic miRNA pool consisted of 2.5 fmol of each of 891 non redundant miRNAs sequences and miRControl 3 seqeunces. Hybridization was performed using an automated hybridization machine (a-Hyb, Miltenyi Biotec). Briefly, volume of labelled sample was adjusted to 100 µl with nuclease-free water and 100 µl 2x Hybridization Solution pre-warmed at 42°C was added. After mixing, the solution was pre-heated to 60 °C for 2 min prior to application to the a-Hyb. Microarray processing in the a-Hyb was as follows: incubation in Pre-Hyb Solution for 5 min at 42°C, hybridization with the labeled RNAs for 960 min at 42°C, wash with Wash Buffer I for 1 min at 10°C (2 cycles), and second wash with Wash Buffer II for 1 min at 10°C (2 cycles). Pump speed at all incubations was set to 1 ml/min. Microarrays were blow-dried and scanned with the ScanArray Lite (GSI Lumonics, Watertown, MA) and the Agilent DNA-Microarray Scanner.
|
| 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 two-fold the mean background value (2bkg dataset). Subsequently,the array data was normalized by calculating the median of the miRControl 3 present in the liver and UR sample.
|
| |
|
| 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 |
GPL8440 |
| Series (1) |
| GSE15835 |
Absolute quantification of miRNAs |
|
