Abstract 1953: Rapid quantitation of circulating microRNAs using a novel detection technology.

Abstract

Abstract The rapid implementation of quantifying microRNAs (miRNAs) in body fluids for diagnosis and prognosis could be improved by designing more rapid approaches. The current mainstream approach is quantitative reverse transcriptase PCR (qRT-PCR). Barriers for the applicability are lengthy time requirement for isolation and amplification of miRNAs, and polymerase inhibitors that copurify with miRNA, and compromise the accuracy of the approach. Here we provide an approach using a nano-particle technology that overcomes these limitations. This technology is based on the principle that a fluorophore-containing oligonucletide sequence is fluorescently silent unless displaced from a quencher by a specific substrate RNA. Once displacement occurs, the resulting fluorescence provides a measure of RNA concentration in the reaction. Our approach allows for the direct and rapid quantification of circulating miRNAs with minimal processing of blood plasma/serum and is done without the use of enzymes. Using circulating miRNAs with established roles in cancer and quality control, miR-16, miR-21 and miR-22 and miR-451, we identified optimal temperature, detergent and solvent-extraction parameters, to quantify accurately these miRNAs in plasma using a microplate fluorometer within an hour after plasma preparation. The stringency of our conditions can discriminate wild type from a single-basepair mismatch RNA, and is specific for mature miRNAs. Furthermore, this approach facilitates measurement of samples collected in heparin, an anticoagulant that severely inhibits polymerases used for qRT-PCR. This approach creates the possibility for rapid blood tests to quantify miRNA-associated events in real-time, and provides greater public access to miRNA studies by reducing the cost and time requirement of current accurate approaches of miRNA quantitation. Citation Format: Dominik Duelli, Jaime Palma, Don Weldon, Justin Doong, Grace Johnston, Fellars Stacey, Michelle Hastings. Rapid quantitation of circulating microRNAs using a novel detection technology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1953. doi:10.1158/1538-7445.AM2013-1953