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.

我们的目标是开发一种新的方法来直接检测血清中的miRNAs， 血浆，而不需要分离总RNA。microRNA（miRNAs）的独特表达谱 最近与癌症和其他疾病有关，这意味着miRNAs可以作为诊断 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检测， 改善治疗结果，并有助于监测进展和对治疗的反应。的RT-qPCR 目前用于定量循环miRNA的测定受到与其表达相关的问题的阻碍。 需要在miRNA定量之前从血浆或血清中分离总RNA。RNA通常 通过旋转柱纯化或苯酚萃取和乙醇沉淀分离。这两种方法都存在以下问题： 不一致的miRNA回收和缺乏用于数据标准化的内部参考miRNA。此外，委员会认为， 目前的RNA分离方法不能完全去除随后RT中使用的酶的抑制剂 PCR反应。因此，不能简单地通过浓缩RNA制剂来提高检测水平 的分析。由于这些问题，目前的RT-qPCR方法最适合于定量 高丰度循环miRNA。然而，许多通过深度测序鉴定的miRNAs发生在 血液样本的浓度太低，无法通过当前的RT-qPCR方法可靠地检测。由此可见，有一 需要一种方法，通过该方法可以直接定量血浆或血清样品中的miRNA，而无需预先RNA 隔离直接的miRNA采样允许数据标准化到样品体积，并改善了两种方法。 RT-qPCR测定的准确性和灵敏度。然而，它需要强大的方法来分离miRNAs， 脂质/蛋白质复合物。在这里，我们提出了一种新的方法，miR-Direct，它提供了直接检测miRNAs 并允许通过捕获和富集miRNA使用更大的样品体积 在检测之前。在初步研究中，我们已经证明了血浆miRNAs的富集 显著提高了TaqMan（R）检测和SomaGenics自己的miR-ID（R）检测的灵敏度 比色法在第一阶段，我们将开发和优化从血浆复合物中释放miRNA的方法， 随后捕获那些感兴趣的miRNA。我们将比较SomaGenics的miR-ID检测的灵敏度， 与TaqMan miRNA检测定量捕获的miRNA，并与标准检测进行比较 对来自血浆的纯化的总RNA进行。然后，使用十种不同的血浆样本，我们将测试 对一组10种与各种癌症相关的miRNA进行优化测定， 血浆中的丰度。我们希望证明miR-Direct可以定量低丰度的miRNAs， 目前的RT-qPCR检测方法无法可靠检测。在第二阶段，我们将使用优化的miR-Direct方法 验证特定癌症类型的miRNA生物标志物候选物，并开发诊断试剂盒 癌症，将与领先的诊断提供商合作商业化。