Laboratoire Gulliver, CNRS, ESPCI Paris, PSL Research University, 10 rue Vauquelin, 75005 Paris, France.
Centre de Recherche des Cordeliers, INSERM, CNRS, Sorbonne Université, USPC, Université Paris Descartes, Université Paris Diderot, Equipe Labellisée Ligue Nationale Contre le Cancer, Paris, France.
Sci Adv. 2020 Jan 22;6(4):eaay5952. doi: 10.1126/sciadv.aay5952. eCollection 2020 Jan.
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.
微 RNA 是一类参与基因表达调控的转录本，它们作为组织或体液中可获取的疾病特异性生物标志物正在逐渐兴起。然而，从生物样本中对其进行准确的定量仍然具有挑战性。我们报告了一种使用等温扩增化学并适应液滴数字读出的敏感和定量微 RNA 检测方法。基于分子编程的概念，我们设计了一种 DNA 电路，该电路可以转换、门限、放大并报告特定微 RNA 的存在，检测下限低至飞摩尔浓度。通过使用泄漏吸收机制，我们能够抑制通常在其他指数扩增反应中遇到的非特异性扩增。结果表明，这种等温扩增方案适用于微 RNA 的数字计数：通过将反应混合物分成油包水液滴，从而实现单个微 RNA 的封装和扩增，该方法提供了绝对的靶标定量。我们的方法的模块化使其能够重新用于各种微 RNA 序列的检测。
