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Isothermal digital detection of microRNAs using background-free molecular circuit

Abstract : MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.
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Contributor : Valerie Taly <>
Submitted on : Friday, November 6, 2020 - 2:49:19 PM
Last modification on : Saturday, December 12, 2020 - 3:58:46 AM

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Guillaume Gines, Roberta Menezes, Kaori Nara, Anne-Sophie Kirstetter, Valérie Taly, et al.. Isothermal digital detection of microRNAs using background-free molecular circuit. Science Advances , American Association for the Advancement of Science (AAAS), 2020, 6 (4), pp.eaay5952. ⟨10.1126/sciadv.aay5952⟩. ⟨inserm-02992640⟩



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