1. Isothermal digital detection of microRNAs using background-free molecular circuit
- Author
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Gines, Guillaume, Menezes, Roberta, Nara, Kaori, Kirstetter, Anne-Sophie, Taly, Valérie, Rondelez, Yannick, Gulliver (UMR 7083), Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), rondelez, yannick, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité)
- Subjects
[SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM] ,[SDV]Life Sciences [q-bio] ,technology, industry, and agriculture ,Reproducibility of Results ,SciAdv r-articles ,Life Sciences ,[SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM] ,Sensitivity and Specificity ,MicroRNAs ,Humans ,Nucleic Acid Amplification Techniques ,Molecular Biology ,Research Articles ,Research Article - Abstract
We report the isothermal digital detection of microRNAs using DNA circuits encoding a background-free amplification mechanism., 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.
- Published
- 2019