Reference Profiles of ExRNA in Biofluids from Well-Defined Human Cohorts

来自明确定义的人类群体的生物体液中 ExRNA 的参考图谱

• 批准号: 9125467
• 负责人: MUNEESH TEWARI
• 金额: $ 7.26万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9125467
• 关键词: Address; Area; Bioinformatics; Biological; Biological Markers; Biological Process; Biology; Blood; Blood Circulation; Blood Tests; Body Fluids; Cells; Clinical; Collection; Conflict (Psychology); Data; Diagnostic tests; Diet; Disease; Electron Microscopy; Encapsulated; Environment; Epidemiologist; Epidemiology; Fractionation; Gene Expression; Genomics; Gold; Health; High-Throughput Nucleotide Sequencing; Hormones; Human; Human Biology; Human body; Immune; Individual; Insecta; Knowledge; Libraries; Liquid substance; Mammals; Mediating; Metadata; Methods; MicroRNAs; Nematoda; Organism; Participant; Pattern; Physiology; Plants; Plasma; Population; Preparation; Production; Protein Binding; Proteins; Protocols documentation; Quality Control; RNA; RNA Sequences; Reference Values; Reporting; Research; Research Infrastructure; Resources; Rice; Sampling; Seminal fluid; Serum; Services; Specimen; Technology; Therapeutic; Time; Transcript; Untranslated RNA; Urine; Validation; Variant; Vascular System; Vegetables; Vesicle; base; cohort; data management; data sharing; design; digital; disease diagnosis; epidemiology study; experience; extracellular; feeding; fruits and vegetables; healthy volunteer; human data; interest; men; microbiome; minimally invasive; nanoparticle; next generation sequencing; novel; repository; research study; sample collection; therapy development; tool; transcriptome sequencing

DESCRIPTION (provided by applicant): The discovery of extracellular RNAs (exRNAs) in the bloodstream has raised intriguing questions about whether regulatory RNA molecules, either endogenously produced or taken up from our environment (e.g., through diet), can influence our physiology by acting as blood-borne hormones. Although we and others have studied circulating exRNAs for several years now, the identity of >95% of exRNA species in blood and other body fluids remains a mystery. Furthermore, although there is evidence that specific diet-derived microRNA (e.g., from rice) can be found in blood, more systematic and comprehensive characterization of blood exRNA is needed to determine the extent to which environmentally-derived RNAs are present in the human circulation. To characterize the spectrum of endogenously and exogenously-derived exRNAs present in human body fluids, we will analyze unique cohorts of well-defined specimens including: (i) well-characterized healthy cohorts of matched plasma, urine and serum from an observational epidemiology study with careful participant selection and well-controlled collection of samples and metadata, (ii) serial plasma samples collected before and after controlled feeding of vegetables in a study of healthy volunteers, and (iii) a cohort of well- characterized semen specimens from healthy men. We have assembled a team of experts to carry out this study including an experienced epidemiologist who has directly overseen collection of all of the plasma, urine and serum specimens, internationally-renowned expertise in the biology of extracellular RNAs in human body fluids, expertise in comprehensive quality assessment of specimens and quality-controlled purification of both extracellular vesicle-encapsulated and protein-bound forms of exRNA, and production-quality expertise in high- throughput sequencing and associated RNA bioinformatics and statistical analysis. We will perform quality assessment, fractionation of vesicle and non-vesicle-associated fractions, and high-throughput sequencing using multiple library preparation to capture as vast a space as possible of exRNAs in the banked human specimens mentioned above. We will use a bioinformatic pipeline to annotate both endogenous and potential exogenously-derived exRNAs, using the design of the controlled vegetable feeding study as a powerful tool to unambiguously assign exogenous origin to specific plant-derived RNAs. Finally, we will also use cutting-edge droplet digital PCR technology to validate and provide absolute quantification of exRNAs of interest, which is essential for establishing reference ranges describing variation in the healthy population.

描述（由申请人提供）： 血流中细胞外RNA（exRNA）的发现提出了一个有趣的问题，即调节RNA分子，无论是内源性产生的还是从我们的环境中摄取的（例如，通过饮食），可以通过血液传播的激素影响我们的生理。尽管我们和其他人已经研究了循环exRNA多年，但血液和其他体液中>95%的exRNA种类的身份仍然是一个谜。此外，尽管有证据表明特定饮食来源的微小RNA（例如，来自水稻）可以在血液中发现，因此需要对血液exRNA进行更系统、更全面的表征，以确定环境来源的RNA在人体循环中的存在程度。 为了表征人体体液中存在的内源性和外源性exRNA的谱，我们将分析明确定义的标本的独特队列，包括：（i）来自观察性流行病学研究的匹配血浆、尿液和血清的良好表征的健康群组，其中仔细选择参与者并良好控制样品和元数据的收集，（ii）在一项健康志愿者研究中，在控制进食蔬菜之前和之后收集的系列血浆样本，以及（iii）来自健康男性的一组特征良好的精液样本。我们组建了一个专家团队来进行这项研究，其中包括一位经验丰富的流行病学家，他直接监督所有血浆、尿液和血清标本的收集，在人体体液中细胞外RNA生物学方面的国际知名专业知识，在标本综合质量评估和细胞外囊泡封装和蛋白结合形式exRNA的质量控制纯化方面的专业知识，以及高通量测序和相关RNA生物信息学和统计分析方面的生产质量专业知识。 我们将进行质量评估，分离囊泡和非囊泡相关组分，并使用多个文库制备进行高通量测序，以在上述库存人类标本中捕获尽可能大的exRNA空间。我们将使用生物信息学管道来注释内源性和潜在的外源来源的exRNA，使用受控蔬菜饲养研究的设计作为一个强大的工具来明确地将外源来源分配给特定的植物来源的RNA。最后，我们还将使用尖端的液滴数字PCR技术来验证和提供感兴趣的exRNA的绝对定量，这对于建立描述健康人群变异的参考范围至关重要。