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.

我们本提案的目标是开发一种直接检测血清和血清中 miRNA 的新方法。 血浆，无需分离总 RNA。 microRNA (miRNA) 的独特表达谱具有 最近发现 miRNA 与癌症和其他疾病有关，这意味着 miRNA 可以作为诊断手段 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，从而导致 改善治疗结果，并有助于监测进展和治疗反应。逆转录定量PCR 目前用于量化循环 miRNA 的检测方法因与其相关的问题而受到阻碍。 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 miRNA 回收率不一致且缺乏用于数据标准化的内部参考 miRNA。而且， 目前的RNA分离程序并不能完全去除后续RT中使用的酶抑制剂 和PCR反应。因此，RNA制剂不能简单地浓缩来提高检测水平 的测定。由于这些问题，当前的 RT-qPCR 方法最适合定量 高丰度循环 miRNA。然而，通过深度测序鉴定出的许多 miRNA 发生在 血液样本浓度太低，目前的 RT-qPCR 方法无法可靠检测。因此，有一个 需要一种可以直接定量血浆或血清样品中 miRNA 的方法，无需事先进行 RNA 隔离。直接 miRNA 采样允许数据标准化为样本量，并提高了 RT-qPCR 检测的准确性和灵敏度。然而，它需要稳健的方法来分离 miRNA 脂质/蛋白质复合物。在这里，我们提出了一种新方法，miR-Direct，可以直接检测 miRNA 从血浆样本中提取，并通过捕获和富集 miRNA 允许使用更大的样本量 检测前感兴趣。在初步研究中，我们已经证明血浆 miRNA 的富集 显着提高 TaqMan(R) 检测和 SomaGenics 自己的 miR-ID(R) 检测的灵敏度 化验。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 使用 TaqMan miRNA 测定法对捕获的 miRNA 进行定量，并将两者与标准测定法进行比较 对从血浆中纯化的总 RNA 进行。然后，我们将使用十种不同的血浆样本来测试 对一组 10 个 miRNA 进行优化测定，这些 miRNA 与各种癌症相关，并且存在于不同的环境中。 血浆中的丰度。我们期望证明 miR-Direct 可以量化低丰度 miRNA， 目前的 RT-qPCR 检测无法可靠地检测到。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为此开发诊断测试套件 癌症，将与领先的诊断提供商合作进行商业化。