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| Status |
Public on Oct 27, 2009 |
| Title |
Absolute quantification of miRNAs |
| Platform organisms |
Homo sapiens; Mus musculus; Rattus norvegicus |
| Sample organisms |
Mus musculus; synthetic construct |
| Experiment type |
Non-coding RNA profiling by array
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| Summary |
MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells.
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| Overall design |
Total liver 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 (n = 6). The synthetic miRNA pool consisted of 2.5 fmol of each of 891 non redundant miRNAs sequences and miRControl 3 sequences.
The array data was normalized by calculating the median of the miRControl 3 present in the liver and UR sample. The miRNA amount was calculated with respect to the corresponding miRNA in the UR.
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| Citation(s) |
19861428 |
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| Submission date |
Apr 27, 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 |
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| Platforms (2) |
| GPL8440 |
miRXploreTM microarrays (856) |
| GPL8441 |
miRXploreTM microarrays (872) |
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| Samples (6)
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| GSM397610 |
liver and spike ins vs. synthetic miRNA pool (1) |
| GSM397611 |
liver and spike ins vs. synthetic miRNA pool (2) |
| GSM397612 |
liver and spike ins vs. synthetic miRNA pool (3) |
| GSM397613 |
liver and spike ins vs. synthetic miRNA pool (4) |
| GSM397614 |
liver and spike ins vs. synthetic miRNA pool (5) |
| GSM397625 |
liver and spike ins vs. synthetic miRNA pool (6) |
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| Relations |
| BioProject |
PRJNA116765 |
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