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Highly Sensitive and Rapid Mix-and-Measure Detection Method for microRNAs in a Ce

Ce 中 microRNA 的高灵敏快速混合和测量检测方法

基本信息

批准号：
7362925
负责人：
Sapna K Deo
金额：
$ 7.39万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-01 至 2009-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7362925
关键词：
Animals Apoptosis Binding Biological Assay Biological Markers Biology Bioluminescence Biomedical Research Breast Cancer Cell Calibration Cell Differentiation process Cell Extracts Cell physiology Chemicals Class Clinical Collaborations Complement Complex Cytoskeleton DNA Data Detection Development Diagnosis Diagnostic Diagnostics Research Disease Disease Progression Enzymes Estrogens Explosion Exposure to Gene Expression Generations Genes Genetic Engineering Goals Health Human Human Genome In Vitro Indiana University Cancer Center Investigation Label Laboratories Luciferases MCF7 cell Malignant Neoplasms Measurement Measures Methods MicroRNAs Mission None or Not Applicable Northern Blotting Nucleotides Oligonucleotide Probes Oligonucleotides One-Step dentin bonding system Outcomes Research Pathogenesis Phase Plants Polymerase Chain Reaction Prognostic Marker Protocols documentation Reaction Renilla Luciferases Reporter Reproducibility Research Research Personnel Role Sampling Signal Transduction Small Interfering RNA Small RNA Solid System Techniques Testing Therapeutic Intervention Time Validation Virus Diseases Work base design desire disorder prevention innovation interest lipid metabolism malignant breast neoplasm nervous system disorder novel strategies prevent research study tool

项目摘要

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are the focus of current biomedical research from the perspective of its role in pathogenesis, diagnosis, and therapeutic intervention of several disease states. But the lack of method that is rapid, quantitative, sensitive, and tolerant to matrix effects imposes limit on measurement of miRNAs in clinical samples and in biomedical research. Our long term goal is to develop rapid, quantitative, and highly sensitive tests for the detection of a panel of miRNA targets in cellular matrix that are vital in biomedical research and disease diagnosis. The objective of this application is to develop highly sensitive one-step detection method for target miRNAs in a cellular extract using split-bioluminescent reporter enzyme followed by demonstrating the application of the strategy in fundamental biomedical research. The central hypothesis of the application is that the formation of an active enzyme from the split enzyme fragments conjugated to oligonucleotide probes upon hybridization provides for the development of mix-and-measure assay, and the use of bioluminescent enzyme allows for direct detection in a cellular extract because there is no background signal associated with bioluminescence signal detection. The rationale for this project is that its successful completion is expected to provide a highly sensitive, quantitative, and rapid one-step method for the detection of any desired miRNA target. Thus the proposed research is relevant to the NIH's mission that pertains to developing sensing of biomolecules that perform essential function in human health. Our hypothesis will be tested by pursuing two specific aims, 1) Design and development of sensitive, mix-and-measure assay method for miR21, miR493, and miR511, 2) Determination of alteration in the levels of target miRNAs in a cellular extract of MCF-7 breast cancer cells exposed to estrogen. The proposed work is innovative, because it introduces a new approach for the detection of microRNA in complex matrix such as cellular extract capitalizing on the use of highly sensitive bioluminescent detection system. The proposed research is significant, because it is expected to provide a sensitive and quantitative tool for the measurement of miRNA that will have an impact on the diagnostics and research performed toward understanding the role of miRNA in human health. MicroRNAs (miRNA) have been found to regulate multiple genes associated with diverse cellular functions such as apoptosis, cell differentiation, fat metabolism, and early development. The role of miRNA in the disease progression is now being heavily investigated from the perspective of pathogenesis, diagnosis, and disease prevention. In that regard, the proposed work is important because it will provide tools for such investigations to be performed in a rapid and sensitive manner. The proposed research is expected to provide an excellent research and diagnostic tool that is essential in investigating the impact of microRNAs on human health.

描述（由申请人提供）：从其在几种疾病状态的发病机制、诊断和治疗干预中的作用的角度来看，MicroRNA（miRNAs）是当前生物医学研究的焦点。但由于缺乏快速、定量、灵敏和耐受基质效应的方法，限制了临床样品和生物医学研究中miRNA的测量。我们的长期目标是开发快速，定量和高灵敏度的检测方法，用于检测细胞基质中的一组miRNA靶点，这些靶点在生物医学研究和疾病诊断中至关重要。本申请的目的是开发使用分裂生物发光报告酶的细胞提取物中靶miRNA的高灵敏度一步检测方法，随后展示该策略在基础生物医学研究中的应用。本申请的中心假设是，在杂交时从缀合至寡核苷酸探针的裂解酶片段形成活性酶提供了混合和测量测定的发展，并且生物发光酶的使用允许在细胞提取物中直接检测，因为不存在与生物发光信号检测相关的背景信号。该项目的基本原理是，它的成功完成预计将提供一种高度灵敏，定量和快速的一步方法，用于检测任何所需的miRNA靶标。因此，拟议中的研究与NIH的使命有关，该使命涉及开发对人类健康起重要作用的生物分子的传感。我们的假设将通过追求两个具体目标来检验，1）设计和开发用于miR 21、miR 493和miR 511的灵敏的混合测量测定方法，2）测定暴露于雌激素的MCF-7乳腺癌细胞的细胞提取物中靶miRNA水平的改变。所提出的工作是创新的，因为它引入了一种新的方法来检测复杂基质中的microRNA，如细胞提取物，利用高灵敏度的生物发光检测系统。这项研究意义重大，因为它有望为测量miRNA提供一种灵敏和定量的工具，这将对理解miRNA在人类健康中的作用的诊断和研究产生影响。 microRNA（miRNA）被发现调控与细胞凋亡、细胞分化、脂肪代谢和早期发育等多种细胞功能相关的多个基因。miRNA在疾病进展中的作用正在从发病机制、诊断和疾病预防的角度进行大量研究。在这方面，拟议的工作很重要，因为它将为以迅速和敏感的方式进行这种调查提供工具。这项研究有望提供一个优秀的研究和诊断工具，这对于研究microRNA对人类健康的影响至关重要。