A new method for multiplex detection of micro-RNAs (miR-ID)

一种多重检测 micro-RNA (miR-ID) 的新方法

• 批准号: 7612777
• 负责人: SERGEI A KAZAKOV
• 金额: $ 30.06万
• 依托单位: SOMAGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-19 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7612777
• 关键词: Affect; Affinity; American; Back; Binding; Biological Assay; Biological Markers; Biological Models; Biological Process; Calibration; Cancer Detection; Cancer Etiology; Cell Cycle; Cell Extracts; Cell Proliferation; Cells; Cessation of life; Chemicals; Chimera organism; Clinical; Compatible; Complement; Complementary DNA; Complex; Condition; DNA; DNA Probes; DNA Sequence; DNA-Directed DNA Polymerase; Data; Detection; Development; Diagnostic; Disease; Disease Progression; Drug Delivery Systems; Early Diagnosis; Elements; Enzymes; Equipment; Figs - dietary; Formalin; Functional RNA; Genes; Goals; Human; Human Genome; Immobilization; Indium; Jurkat Cells; Label; Ligation; Link; Malignant neoplasm of prostate; Masks; Methods; MicroRNAs; Modeling; Modification; Molecular Profiling; Monitor; Noise; Nucleic Acid Probes; Nucleic Acids; Nucleotides; Numbers; Pan Genus; Paraffin Embedding; Pattern; Peptide Signal Sequences; Phase; Play; Polymerase Chain Reaction; Positioning Attribute; Primer Extension; Procedures; Process; Production; Prostate; Prostate carcinoma; Proteins; Public Health; Publishing; RNA; RNA Degradation; RNA Polymerase II; RNA Probes; RNA Sequences; RNA analysis; Range; Reagent; Regulation; Reporting; Research; Reverse Transcription; Sampling; Scheme; Sensitivity and Specificity; Signal Transduction; Slide; Small RNA; Solutions; Specificity; Standards of Weights and Measures; Structure; Surface; Techniques; Temperature; Testing; Time; Tissues; Translations; Variant; Vendor; Viral Cancer; Virus Diseases; Work; Zip Code; base; cancer type; cost; crosslink; design; high throughput screening; human DICER1 protein; improved; magnetic beads; men; multiplex detection; novel; novel strategies; nuclease; pre-miRNA; prevent; prognostic; response; sample fixation; synthetic construct; therapeutic target; tumor progression

DESCRIPTION (provided by applicant): A new method for multiplex detection of micro-RNAs (miR-ID) ABSRACT MicroRNAs (miRNAs) and other short non-coding RNAs play diverse biological functions, and distinctive miRNA expression patterns are associated with various types of cancer and viral infections. Thus, miRNAs may be considered as potential diagnostic biomarkers as well as potential drug targets. Many different methods for detecting miRNAs and profiling their expression patterns have been developed recently. However, none of these methods is capable of quantifying different miRNAs with equal accuracy and sensitivity, and all suffer from problems such as laborious handling and purification steps, and expensive equipment and reagents. Here we propose a novel method, which we call miR-ID, that allows fast, accurate, sensitive, and highly multiplexable quantification of short target RNAs such as miRNAs through signal (rather than target) amplification. The miR-ID approach is also potentially less complex and expensive than alternative available methods. MiR-ID employs a probe that contains a miRNA-specific capture sequence as well as signal sequences. This probe is expected to efficiently and sequence-specifically capture target miRNAs directly from nucleic acid extracts or cell lysates. The probe-miRNA complex is then processed in such a way that it can be quantified by qPCR. The proposed detection method has no need for chemical or enzymatic labeling of miRNAs, avoids sequence-dependent bias that could be introduced by miRNA ligation and reverse transcription, is compatible with qPCR machines from a variety of vendors, and allows massive multiplexing. In Phase I, we will test several different designs of capture probes and seek the best combination of sensitivity, sequence-specificity, low cost, and workflow time. We will also verify the sensitivity and specificity of the method in a "real-world" setting by quantifying cellular miRNAs and comparing our results with published data obtained with other methods. In Phase II, we plan to develop a diagnostic assay for prostate cancer detection as well as for monitoring progression of the disease and response to therapy. PUBLIC HEALTH RELEVANCE: Prostate cancer is the second leading cause of cancer deaths among American men. The goal of this application is early diagnosis of this disease based on quantification of a panel of microRNAs that constitutes a promising biomarker. The method of micro-RNA detection has novel features that promise better sensitivity and selectivity than other micro-RNA assays currently available.

描述（由申请人提供）：一种用于微RNA（miR-id）弃and microRNA（miRNA）和其他短的非编码RNA的新方法，具有多种生物学功能，独特的miRNA表达模式与各种类型的癌症和病毒感染有关。因此，miRNA可以被视为潜在的诊断生物标志物以及潜在的药物靶标。最近已经开发出许多用于检测miRNA和分析其表达模式的不同方法。但是，这些方法都无法以平等的精度和敏感性来量化不同的miRNA，并且所有这些方法都遭受了诸如费力的处理和纯化步骤以及昂贵的设备和试剂等问题。在这里，我们提出了一种新的方法，我们称之为miR-id，它可以通过信号（而不是目标）放大对短目标RNA（例如miRNA）等快速，准确，敏感和高度多重的量化。与替代性可用方法相比，miR-ID方法的复杂和昂贵。 miR-ID采用包含miRNA特异性捕获序列和信号序列的探针。该探针有望直接从核酸提取物或细胞裂解物中直接捕获靶miRNA，并特异性地捕获该探针。然后，处理探针 - miRNA复合物的处理方式，以使其可以通过qPCR进行量化。所提出的检测方法不需要miRNA的化学或酶促标记，避免了miRNA连接和逆转录可能引入的序列依赖性偏差，与来自多种供应商的qPCR机器兼容，并允许大量的多路复用。在第一阶段，我们将测试几种不同的捕获探针设计，并寻求灵敏度，序列特异性，低成本和工作流时间的最佳组合。我们还将通过量化细胞miRNA并将我们的结果与其他方法获得的已公开数据进行比较，从而在“现实世界”设置中验证该方法的敏感性和特异性。在第二阶段，我们计划开发用于前列腺癌检测的诊断测定法，以及监测疾病进展和对治疗的反应。公共卫生相关性：前列腺癌是美国男性癌症死亡的第二大原因。该应用的目的是基于对构成有希望的生物标志物的一群microRNA的量化，对这种疾病的早期诊断。微RNA检测方法具有新的特征，比当前可用的其他微RNA分析有望更好地敏感性和选择性。