Absolute quantification of microRNAs by using a universal reference Export

@article{citeulike:6038935, abstract = {10.1261/rna.1754109 MicroRNAs (miRNAs) are a species of small RNAs \^{a}ˆ¼21\^{a}€“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 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 the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/CD133(\^{a}ˆ’) hematopoietic progenitor cells.}, author = {Bissels, Ute and Wild, Stefan and Tomiuk, Stefan and Holste, Angela and Hafner, Markus and Tuschl, Thomas and Bosio, Andreas}, citeulike-article-id = {6038935}, citeulike-linkout-0 = {http://dx.doi.org/10.1261/rna.1754109}, citeulike-linkout-1 = {http://rnajournal.cshlp.org/content/15/12/2375.abstract}, citeulike-linkout-2 = {http://rnajournal.cshlp.org/content/15/12/2375.full.pdf}, citeulike-linkout-3 = {http://view.ncbi.nlm.nih.gov/pubmed/19861428}, citeulike-linkout-4 = {http://www.hubmed.org/display.cgi?uids=19861428}, day = {27}, doi = {10.1261/rna.1754109}, issn = {1469-9001}, journal = {RNA}, keywords = {mechanism, methods, mirna}, month = {December}, number = {12}, pages = {2375--2384}, posted-at = {2009-10-30 01:37:50}, priority = {2}, title = {Absolute quantification of microRNAs by using a universal reference}, url = {http://dx.doi.org/10.1261/rna.1754109}, volume = {15}, year = {2009} }

TY - JOUR ID - citeulike:6038935 L3 - citeulike-article-id:6038935 N2 - 10.1261/rna.1754109 MicroRNAs (miRNAs) are a species of small RNAs ∼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 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 the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/CD133(−) hematopoietic progenitor cells. IS - 12 JF - RNA SN - 1469-9001 EP - 2384 TI - Absolute quantification of microRNAs by using a universal reference VL - 15 SP - 2375 KW - mechanism KW - methods KW - mirna AU - Bissels, Ute AU - Wild, Stefan AU - Tomiuk, Stefan AU - Holste, Angela AU - Hafner, Markus AU - Tuschl, Thomas AU - Bosio, Andreas PY - 2009/12/27/ UR - http://dx.doi.org/10.1261/rna.1754109 ER -