P.R.I.S.M: Purification of exRNA by Immuno-capture and Sorting using Microfluidic

摘要 细胞外RNA(ExRNAs)由一系列不同的载体保护，在生物体液中不被降解，目前 被认为是很有前途的生物标志物。事实上，研究表明，在疾病发展过程中， 细胞/组织修改它们由一个或几个蛋白质或囊泡载体携带的分泌型exRNA的注册表， 然后参与修改相关生物体液中的全球exRNAs配置文件。然而，临床上- 相关的exRNA修饰通常代表给定生物体液中循环的exRNA的一小部分， 然后在检查整个生物体液exRNAs图谱时可能保持检测不到。因此，建立 有效和特定的exRNAs-载体分离方法，以及下游相关的exRNAs参考图谱 在正常和疾病情况下，是实施生物流体exRNAs分析的先决条件 作为常规诊断工具。 P.R.I.S.M项目(免疫捕获和微流控分离纯化外源RNA)的目标， 是将粘解性细胞外纳米胶囊分选与蛋白质亲和力捕获方法相结合，使用不同的 微流控芯片设备，以分离不同的exRNA载体(囊泡、脂蛋白或RNA结合 从单个人体体液样本(血浆、脑脊液、尿液、牛奶)中提取蛋白质)，然后进行RNA分析。这个项目 不仅将为不同的人体体液建立必要的载体特有的exRNA参考图谱，而且应该 也大大提高了基于exRNA的诊断分析的重复性、速度、敏感性和特异性 与目前该领域最先进的技术相比。 相关性 生物体液中循环的细胞外RNA(ExRNAs)受到不同蛋白质和囊泡载体的保护 目前被认为是疾病的有希望的生物标记物，比如癌症。然而，只有一小部分人 总exRNA聚集在某些特定的exRNA载体中，包含临床相关信息。我们的 该项目旨在开发微流控方法，将生物流体分馏成最相关的exRNAs载体 在确定它们的exRNA含量之前，从单个小容量的生物流体样本中提取成分。vt.在.的基础上 结论：我们期望在exRNA诊断的特异性和敏感性方面有显著的提高。 战略。

SUMMARY Extracellular RNAs (exRNAs), protected from degradation in biofluids by a diverse set of carriers, are currently considered as promising biomarkers. Indeed, it was shown that during disease progression, impacted cells/tissues modify their registries of secreted exRNAs, carried by one or several protein or vesicular carriers, which then participate in a modification of the global exRNAs profile in related biofluids. However, the clinically- relevant exRNA modifications often represent a small fraction of the circulating exRNA within a given biofluid, and could then remain undetectable when examining total biofluids exRNAs profiles. As such, establishing efficient and specific exRNAs-carrier isolation methods, and downstream associated exRNAs reference profiles in normal and disease situations, represents a prerequisite towards implementation of biofluid exRNAs profiling as a routine diagnostic tool. The objective of the P.R.I.S.M project (Purification of exRNA by Immuno-capture and Sorting using Microfluidics), is to combine viscoleastic extracellular nanovesicle sorting with protein-affinity capture methods, using different microfluidic chip device, in order to isolate distinct exRNA carriers (vesicles, lipoproteins, or RNA-binding proteins) from a single sample of human biofluids (plasma, CSF, urine, milk), prior to RNA profiling. This project will not only establish essential carrier-specific exRNA reference profiles for different human biofluids, but should also dramatically improve the reproducibility, speed, sensitivity and specificity of exRNA-based diagnostic assays compared to the current state-of-the-art in the field. RELEVANCE Circulating extracellular RNA (exRNAs) in biofluids, protected by distinct proteins and vesicular carriers, are currently considered as promising biomarkers in diseases, such a cancer. However, only a small percentage of the total exRNAs, clustered in some specific exRNA carriers, contain the clinically relevant information. Our project aims at developing microfluidic methods for fractionating biofluid into its most relevant exRNAs carrier components, from a single low volume of biofluid sample before characterizing their exRNA content. Upon conclusion, we expect to reach a dramatic improvement in the specificity and sensitivity of exRNA diagnostic strategies.