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 目前，细胞外 RNA (exRNA) 受到多种载体的保护，免于在生物体液中降解。 被认为是有前途的生物标志物。事实上，研究表明，在疾病进展过程中， 细胞/组织修改其分泌的exRNA的登记，由一种或多种蛋白质或囊泡载体携带， 然后参与相关生物流体中整体 exRNA 谱的修改。然而，临床上—— 相关的 exRNA 修饰通常代表给定生物流体中循环 exRNA 的一小部分， 然后在检查总生物体液 exRNA 谱时仍然无法检测到。因此，建立 高效且特异的 exRNA 载体分离方法，以及下游相关 exRNA 参考资料 在正常和疾病情况下，是实施生物流体 exRNA 分析的先决条件 作为常规诊断工具。 P.R.I.S.M 项目的目标（使用微流体通过免疫捕获和分选纯化 exRNA）， 是将粘性细胞外纳米囊泡分选与蛋白质亲和捕获方法相结合，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 在进行 RNA 分析之前，从人类生物体液的单个样本（血浆、脑脊液、尿液、牛奶）中提取蛋白质）。本项目 不仅会为不同的人类生物体液建立重要的载体特异性 exRNA 参考图谱，而且应该 还显着提高基于 exRNA 的诊断测定的再现性、速度、灵敏度和特异性 与该领域目前的最新技术相比。 关联 生物体液中的循环细胞外 RNA (exRNA) 受到不同蛋白质和囊泡载体的保护， 目前被认为是癌症等疾病的有前途的生物标志物。然而，只有一小部分人 聚集在一些特定 exRNA 载体中的总 exRNA 包含临床相关信息。我们的 该项目旨在开发微流体方法，将生物流体分级为其最相关的 exRNA 载体 在表征其 exRNA 含量之前，从单个低体积的生物流体样品中提取成分。之上 结论是，我们期望 exRNA 诊断的特异性和敏感性得到显着提高 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.