Ligo-miR - A Multiplexed Single Molecule Ligation Assay for miRNA Profiling

Ligo-miR - 用于 miRNA 分析的多重单分子连接测定

• 批准号: 8550117
• 负责人: Kelvin Liu
• 金额: $ 19.98万
• 依托单位: CIRCULOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-24 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8550117
• 关键词: Architecture; Biological Assay; Biological Markers; Buffers; Cancer cell line; Capillary Electrophoresis; Cell Line; Cell physiology; Cells; Clinical; Complementary DNA; Cytolysis; Data; Detection; Development; Devices; Diagnostic; Dimensions; Disease; Foundations; Functional RNA; Gene Expression; Gene Expression Regulation; Human; Lead; Length; Ligase; Ligation; Malignant Neoplasms; Methods; Methylation; MicroRNAs; Microfluidic Microchips; Microfluidics; Molecular; Molecular Profiling; Mutation Analysis; Neoplasm Circulating Cells; Normal tissue morphology; Nucleic Acid Probes; Nucleic Acids; Oligonucleotides; Phase; Play; Publishing; Quantitative Reverse Transcriptase PCR; RNA; Reaction; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Role; Sampling; Sensitivity and Specificity; Solutions; Techniques; Testing; Tissues; Transcript; Untranslated RNA; Variant; Work; base; cDNA Library; cancer therapy; design; effective therapy; experience; genetic analysis; locked nucleic acid; method development; multiplex detection; nanoparticle; neoplastic cell; pressure; research study; single cell analysis; single molecule; tumor; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): Ligo-miR - A Multiplexed Single Molecule Ligation Assay for miRNA profiling MicroRNAs (miRNA) are short, noncoding RNAs with pervasive roles throughout gene expression in cellular processes such as differentiation and disease states such as cancer. Uncovering the roles of these molecules in development and tumorigenesis are key steps to the discovery of robust, new biomarkers and potential disease cures. The ability to profile miRNA expression at single cell resolution across a tumor mass could lead to targeted therapies that are effective at eliminating rather than merely shrinking tumors. No existing miRNA analysis method combines high sensitivity with true multiplexing and small volume capability. In this Phase I SHIFT proposal, a PCR-free, multiplex ligation assay for miRNA profiling called Ligo-miR will be developed. Hybridization and ligation of locked nucleic acid probes will be used to generate miRNA specific ligation products encoded by length. The ligation products will then be directly identified and quantified using microfluidic single molecul free solution hydrodynamic separation (SML-FSHS). The ligation mechanism will enable Ligo-miR to perform multiplex detection of up to 20 miRNA per reaction while the single molecule analysis platform will enable PCR-free detection with a sensitivity of <20 copies and sample volume <10 pL. This unique combination of high sensitivity and near-zero sample volume will form the foundation for a Phase II single cell miRNA profiling platform. Furthermore, this architecture can be easily scaled to even higher degrees of multiplexing (>50-plex) and throughput through microfluidics. In Aim 1, we will develop the fundamental Ligo-miR assay using synthetic RNA targets to mimic 3 classical miRNAs, let-7a, miR-16, and miR-21. In Aim 2, we will design a SML-FSHS microfluidic device to analyze the ligation products generated in Aim 1. In Aim 3, we will integrate these techniques into a multiplexed assay that can detect 20 miRNAs per reaction. Finally, in Aim 4, we will use the 20-plex assay to profile miRNA in 3 human cancer cell lines and 3 normal tissues. The Ligo-miR results will then be compared to published microarray and RT-PCR data. Such a method not only has applications in miRNA tumor profiling but also in other applications with rare samples such as clinical diagnostics using circulating tumor cells and cell-free miRNA.

描述（由申请人提供）：Ligo -mir- miRNA分析microRNA（miRNA）的多路复用单分子连接测定法是整个基因表达的短而无编码的RNA，在分化和疾病状态等细胞过程中，具有普遍性的作用。揭示这些分子在发育和肿瘤发生中的作用是发现健壮，新生物标志物和潜在疾病治疗方法的关键步骤。跨肿瘤质量的单细胞分辨率miRNA表达的能力可能导致有效消除而不是仅仅缩小肿瘤的靶向疗法。没有现有的miRNA分析方法将高灵敏度与真实的多路复用和较小的体积能力相结合。在这一阶段的偏移提案中，将开发一种无PCR的，无PCR的多重连接测定，用于miRNA分析称为Ligo-MIR。锁定核酸探针的杂交和连接将用于产生按长度编码的miRNA特异性连接产物。然后，连接产物将使用微流体单分子溶液流体动力学分离（SML-FSH）直接识别和定量。连接机制将使Ligo-MIR能够执行每个反应最多20个miRNA的多重检测，而单分子分析平台将启用无PCR检测，灵敏度<20拷贝，样品体积<10 pl。这种高灵敏度和接近零样品体积的独特组合将为II期单细胞miRNA分析平台构成基础。此外，该体系结构可以很容易地缩放到更高的多路复用度（> 50-plex）和通过微流体的吞吐量。在AIM 1中，我们将使用合成的RNA靶标开发基本的Ligo-MIR分析，以模拟3经典miRNA，Let-7a，miR-16和miR-21。在AIM 2中，我们将设计一个SML-FSHS微流体设备来分析AIM 1中生成的连接产物。在AIM 3中，我们将这些技术集成到一个多路复用的测定中，该测定法可以检测每个反应的20个miRNA。最后，在AIM 4中，我们将使用20个PLEX测定法对3种人类癌细胞系和3种正常组织中的miRNA进行介绍。然后将Ligo-MIR结果与已发布的微阵列和RT-PCR数据进行比较。这种方法不仅在miRNA肿瘤分析中应用，而且还具有其他罕见样品（例如使用临床诊断）的应用 循环肿瘤细胞和无细胞miRNA。