Direct RT-qPCR analysis of microRNAs in human plasma (miR-Direct)

人血浆中 microRNA 的直接 RT-qPCR 分析 (miR-Direct)

• 批准号: 8646609
• 负责人: SERGEI A KAZAKOV
• 金额: $ 22.5万
• 依托单位: SOMAGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8646609
• 关键词: Address; Aliquot; Applications Grants; Benchmarking; Biological Assay; Biological Markers; Blood; Blood specimen; Buffers; Cause of Death; Complex; Cytolysis; DNA Probes; Data; Detection; Detergents; Diagnostic; Diagnostic tests; Disease; Dissociation; Encapsulated; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Ethanol; Goals; Heart Diseases; High-Throughput Nucleotide Sequencing; Human; Kinetics; Lipids; Literature; Malignant Neoplasms; Measures; Methods; MicroRNAs; Molecular; Molecular Profiling; Monitor; Outcome; Peptide Hydrolases; Phase; Phenols; Plasma; Precipitation; Preparation; Procedures; Prognostic Marker; Protocols documentation; Provider; RNA; Reaction; Reagent; Recovery; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases; Sampling; Screening for cancer; Second Primary Cancers; Sensitivity and Specificity; Serum; Solutions; Streptavidin; Technology; Testing; Therapeutic; Time; base; cancer type; commercialization; deep sequencing; design; extracellular; improved; inhibitor/antagonist; interest; internal control; magnetic beads; novel; nuclease; protein complex; public health relevance; response; scale up; tool

Our goal in this proposal is to develop a novel method for the direct detection of miRNAs in serum and plasma without requiring isolation of total RNA. Distinct expression profiles of microRNAs (miRNAs) have recently been associated with cancer and other diseases, implying that miRNAs could serve as diagnostic biomarkers for these diseases. Effective biomarkers could facilitate early detection of cancer, leading to improved therapeutic outcomes, and aid in monitoring progression and response to therapy. The RT-qPCR assays currently employed to quantify circulating miRNAs are hampered by problems associated with their requirement for isolating total RNA from plasma or serum prior to miRNA quantification. RNA is typically isolated by spin-column purification or phenol extraction and ethanol precipitation. Both methods suffer from inconsistent miRNA recovery and the lack of an internal reference miRNA for data normalization. Moreover, current RNA isolation procedures do not completely remove inhibitors of enzymes used in the subsequent RT and PCR reactions. Therefore, RNA preparations cannot simply be concentrated to improve the detection level of the assays. As a result of these issues, the current RT-qPCR methods are best suited for quantification of high-abundance circulating miRNAs. However, many miRNAs that were identified by deep sequencing occur in blood samples at concentrations too low to be reliably detected by current RT-qPCR methods. Thus, there is a need for a method by which miRNA in plasma or serum samples can be quantifies directly, without prior RNA isolation. Direct miRNA sampling allows data normalization to the sample volume and improves both the accuracy and sensitivity of RT-qPCR assays. However, it requires robust methods of separating miRNAs from lipid/protein complexes. Here, we propose a new method, miR-Direct, that provides direct detection of miRNAs from plasma samples and allows use of larger sample volumes through the capture and enrichment of miRNAs of interest before detection. In preliminary studies we have demonstrated that enrichment of plasma miRNAs significantly increases the sensitivity of detection by both the TaqMan(R) assay and SomaGenics' own miR-ID(R) assay. In Phase I, we will develop and optimize methods for liberating miRNAs from plasma complexes and subsequent capture of those miRNAs of interest. We will compare the sensitivity of SomaGenics' miR-ID assay with TaqMan miRNA assay in quantifying the captured miRNAs, and compare both with standard assays performed on purified total RNA from plasma. Then, using ten different plasma samples, we will test the optimized assay on a panel of 10 miRNAs that are associated with various cancers and are present at different abundances in plasma. We expect to demonstrate that miR-Direct can quantify low abundance miRNAs that are not reliably detected by current RT-qPCR assays. In Phase II, we will use the optimized miR-Direct method to validate miRNA biomarker candidates for a specific cancer type and develop a diagnostic test kit for this cancer, to be commerciaized in a partnership with a leading diagnostic provider.

我们在此提案中的目标是开发一种新颖的方法，用于直接检测血清中的miRNA和 血浆无需分离总RNA。 microRNA（miRNA）的不同表达曲线具有 最近与癌症和其他疾病有关，这意味着miRNA可以用作诊断 这些疾病的生物标志物。有效的生物标志物可以促进癌症的早期发现，导致 改善治疗结果，并有助于监测治疗的进展和反应。 RT-QPCR 目前用来量化循环miRNA的测定法受到与之相关的问题的阻碍 在miRNA定量之前，从血浆或血清中分离总RNA的要求。 RNA通常是 通过自旋柱纯化或苯酚提取和乙醇沉淀分离。两种方法都遭受 miRNA恢复不一致，缺乏用于数据归一化的内部参考miRNA。而且， 当前的RNA隔离程序不会完全去除随后的RT中使用的酶的抑制剂 和PCR反应。因此，RNA制剂不能简单地集中以提高检测水平 测定。由于这些问题，当前的RT-QPCR方法最适合定量 高丰度循环miRNA。但是，许多通过深度测序发现的miRNA发生在 浓度太低而无法通过当前RT-QPCR方法可靠地检测到的血液样本。因此，有一个 需要一种可以直接量化血浆或血清样品中miRNA的方法，而无需先前的RNA 隔离。直接miRNA采样允许数据归一化到样品体积，并改进了 RT-QPCR分析的准确性和灵敏度。但是，它需要强大的方法将miRNA与 脂质/蛋白质复合物。在这里，我们提出了一种新的方法，即MiR指导，可直接检测miRNA 从血浆样品中，允许通过捕获和富集使用较大的样品体积 检测前感兴趣。在初步研究中，我们证明了血浆miRNA的富集 大大提高了Taqman（R）测定和体体的Mir-ID（R）的检测敏感性 测定。在第一阶段，我们将开发并优化从等离子体复合物中解放miRNA的方法， 随后捕获那些感兴趣的miRNA。我们将比较体体的miR-ID分析的敏感性 用Taqman miRNA测定法量化捕获的miRNA，并将两者与标准测定法进行比较 从血浆中纯化的总RNA进行。然后，使用十个不同的等离子体样本，我们将测试 在一个与各种癌症相关的10个miRNA面板上进行了优化测定，并且存在于不同 血浆中的丰富性。我们希望表明miR指导可以量化低丰度miRNA 当前的RT-QPCR分析无法可靠地检测到。在第二阶段，我们将使用优化的miR指导方法 为特定癌症类型验证miRNA生物标志物候选物，并为此开发诊断测试套件 癌症，与领先的诊断提供者合作伙伴关系。