Transcriptomics,Multiomics

Dataset Information

5

Absolute quantification of miRNAs


ABSTRACT: 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. 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.

SUBMITTER: Ute Bissels  

PROVIDER: E-GEOD-15835 | ArrayExpress | 2010-06-20

SECONDARY ACCESSION(S): GSE15835PRJNA116765

REPOSITORIES: GEO, ArrayExpress

Dataset's files

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Action DRS
E-GEOD-15835.README.txt Txt
E-GEOD-15835.idf.txt Idf
E-GEOD-15835.processed.1.zip Processed
E-GEOD-15835.raw.1.zip Raw
E-GEOD-15835.sdrf.txt Txt
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Publications

Absolute quantification of microRNAs by using a universal reference.

Bissels Ute U   Wild Stefan S   Tomiuk Stefan S   Holste Angela A   Hafner Markus M   Tuschl Thomas T   Bosio Andreas A  

RNA (New York, N.Y.) 20091027 12


MicroRNAs (miRNAs) are a species of small RNAs approximately 21-23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While mi  ...[more]

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