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

总结 细胞外RNA（exRNA），通过一组不同的载体被保护免于在生物流体中降解，目前是 被认为是有前途的生物标志物。事实上，研究表明，在疾病进展期间， 细胞/组织修饰它们的分泌exRNA的登记，所述exRNA由一种或几种蛋白质或囊泡载体携带， 其随后参与相关生物流体中的全局exRNA谱的修饰。但是，临床上- 相关的exRNA修饰通常代表给定生物流体内循环exRNA的一小部分， 然后在检查总生物流体exRNA谱时保持不可检测。因此，建立 高效和特异的exRNA载体分离方法，以及下游相关exRNA参考谱 在正常和疾病情况下，代表了实施生物流体exRNA谱分析的先决条件 作为常规诊断工具。 P.R.I.S. M项目的目的（使用微流体通过免疫捕获和分选纯化exRNA）， 是将联合收割机粘弹性细胞外纳米囊泡分选与蛋白质亲和捕获方法结合，使用不同的 微流控芯片装置，以分离不同的exRNA载体（囊泡、脂蛋白或RNA结合蛋白）。 在RNA谱分析之前，从人生物流体（血浆、CSF、尿、乳）的单个样品中提取蛋白质）。这个项目 将不仅为不同的人类生物流体建立必需的载体特异性exRNA参考谱，而且应该 还显著提高了基于exRNA的诊断测定的再现性、速度、灵敏度和特异性 与目前该领域的最先进技术相比。 相关性 生物流体中的循环细胞外RNA（exRNA）受到不同蛋白质和囊泡载体的保护， 目前被认为是疾病如癌症中有前途的生物标志物。然而，只有一小部分 总的exRNA聚集在一些特定的exRNA载体中，包含临床相关信息。我们 该项目旨在开发微流控方法，将生物流体分离成最相关的exRNAs载体 在表征它们的exRNA含量之前，从单个低体积的生物流体样品中提取组分。后 结论，我们期望在exRNA诊断的特异性和灵敏度上达到显著的提高， 战略布局

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.