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

P.R.I.S.M：使用微流体通过免疫捕获和分选纯化 exRNA

• 批准号: 10490894
• 负责人: Bogdan Mateescu
• 金额: $ 72.58万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10490894
• 关键词: Affinity; Alpha Particles; Antibodies; Antisense Oligonucleotides; Biological Assay; Biological Markers; Blood Platelets; Cell Culture Techniques; Cells; Centrifugation; Chylomicrons; Code; Collection; Custom; Data; Detection; Devices; Disease; Disease Progression; Double-Stranded RNA; Epitopes; Extracellular Protein; Filtration; Fractionation; Gel; High Density Lipoproteins; Human; Immunoprecipitation; Individual; Knock-out; Lateral; Libraries; Lipoprotein Binding; Lipoproteins; Liquid substance; Low-Density Lipoproteins; Malignant Neoplasms; Membrane Proteins; Messenger RNA; Methods; MicroRNAs; Microfluidic Microchips; Microfluidics; Microspheres; Milk; Mining; Modification; Peptide antibodies; Phase; Plasma; Plumbing; Procedures; Process; Proteins; Proteomics; Protocols documentation; Quantitative Reverse Transcriptase PCR; RNA; RNA Sequences; RNA-Binding Proteins; Registries; Reproducibility; Sampling; Sensitivity and Specificity; Series; Small RNA; Sorting - Cell Movement; Speed; System; Testing; Tissues; Transfer RNA; Urine; Validation; Vesicle; Western Blotting; base; clinically relevant; diagnostic assay; diagnostic strategy; diagnostic tool; digital; exosome; extracellular; extracellular vesicles; improved; innovation; member; microvesicles; migration; multiplex assay; nanoparticle; nanovesicle; particle; transcriptome sequencing; viscoelasticity

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.

概括 当前，通过多种载体保护生物流体的降解，细胞外RNA（EXRNA）目前是 被认为是有前途的生物标志物。确实，结果表明，在疾病进展中受到影响 细胞/组织修改其分泌的Exrnas的注册，由一个或几种蛋白质或囊泡载体携带 然后参与相关生物流体中全局EXRNA概况的修改。但是，在临床上 - 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量表时可能保持不可检测。因此，建立 高效，特定的Exrnas载体隔离方法，以及下游相关的Exrnas参考曲线 在正常情况和疾病情况下，代表实施生物流体EXRNA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（通过免疫捕获和使用微流体分类对EXRNA进行纯化）， 是使用不同 微流体芯片装置，以隔离不同的ExRNA载体（囊泡，脂蛋白或RNA结合 RNA分析之前，来自人类生物流体的单个样品（血浆，CSF，尿液，牛奶）的蛋白质）。这个项目 不仅会为不同人类生物流体建立特定于特定的载体EXRNA参考曲线，还应 还显着提高了基于EXRNA的诊断测定的可重复性，速度，灵敏度和特异性 与该领域的当前最新面积相比。 关联 在生物流体中循环的细胞外RNA（EXRNA），受不同的蛋白质和囊泡载体保护的是 目前被认为是疾病中有前途的生物标志物，例如一种癌症。但是，只有一小部分 聚集在某些特定ExRNA载体中的总exrnas包含临床相关信息。我们的 项目旨在开发微流体方法，以将生物流体分馏为最相关的Exrnas载体 成分，来自单个低体积的生物流体样品，然后表征其ExRNA含量。之上 结论，我们期望ExRNA诊断的特异性和灵敏度有了显着提高 策略。

项目成果

Direct isolation of small extracellular vesicles from human blood using viscoelastic microfluidics.

• DOI: 10.1126/sciadv.adi5296
• 发表时间: 2023-10-06
• 期刊: SCIENCE ADVANCES
• 影响因子: 13.6
• 作者: Meng, Yingchao;Zhang, Yanan;Buhler, Marcel;Wang, Shuchen;Asghari, Mohammad;Sturchler, Alessandra;Mateescu, Bogdan;Weiss, Tobias;Stavrakis, Stavros;deMello, Andrew J.
• 通讯作者: deMello, Andrew J.