Isothermal digital detection of microRNA using background-free molecular circuit
| Autor: | Anne-Sophie Kirstetter, Valérie Taly, Yannick Rondelez, Kaori Nara, Guillaume Gines, Roberta Menezes |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: | |
| Zdroj: | BioArXiv bioRxiv |
| DOI: | 10.1101/701276 |
| Popis: | MicroRNA, 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 method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design DNA circuit that converts, threshold, amplifies and report the presence of a specific microRNA, down to the femtomolar concentration. Using a leak-absorption mechanism, we were able to suppress non-specific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNA: 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. |
| Databáze: | OpenAIRE |
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