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Isothermal digital detection of microRNAs using background-free molecular circuit.
Science Advances ( IF 13.6 ) Pub Date : 2020-01-22 , DOI: 10.1126/sciadv.aay5952
Guillaume Gines 1 , Roberta Menezes 2 , Kaori Nara 1 , Anne-Sophie Kirstetter 1 , Valerie Taly 2 , Yannick Rondelez 1
Affiliation  

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.

中文翻译:

使用无背景分子电路的等温数字检测微小RNA。

MicroRNA是一类参与基因表达调控的转录物,正在成为可从组织或体液中获得的有前途的疾病特异性生物标志物。但是,从生物样品中准确定量仍具有挑战性。我们报告了一种敏感的和定量的microRNA检测方法,使用了适合液滴数字读出的等温扩增化学方法。在分子编程概念的基础上,我们设计了一种DNA电路,该电路可以转换,阈值,扩增并报告特定的microRNA(低至飞摩尔浓度)的存在。使用泄漏吸收机制,我们能够抑制非特异性扩增,这在其他指数扩增反应中通常会遇到。结果是,我们证明了这种等温扩增方案适用于microRNA的数字计数:通过将反应混合物分配到油包水小滴中,导致单个microRNA的包封和扩增,该方法可提供绝对的靶标定量。我们方法的模块化使得能够将测定法重新用于各种microRNA序列。
更新日期:2020-01-23
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