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

基本信息

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
项目类别：
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

项目摘要

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 - 用于 miRNA 分析的多重单分子连接测定 MicroRNA (miRNA) 是短的非编码 RNA，在细胞过程（例如分化和疾病状态（例如癌症））的整个基因表达中发挥普遍作用。揭示这些分子在发育和肿瘤发生中的作用是发现强大的新生物标志物和潜在疾病治疗方法的关键步骤。以单细胞分辨率分析整个肿瘤块中 miRNA 表达的能力可能会导致有效消除肿瘤而不仅仅是缩小肿瘤的靶向治疗。现有的 miRNA 分析方法还没有将高灵敏度与真正的多重分析和小体积能力结合起来。在这一 I 期 SHIFT 提案中，将开发一种用于 miRNA 分析的无 PCR 多重连接测定，称为 Ligo-miR。锁核酸探针的杂交和连接将用于产生由长度编码的miRNA特异性连接产物。然后使用微流控单分子自由溶液流体动力学分离（SML-FSHS）直接鉴定和定量连接产物。连接机制将使 Ligo-miR 每个反应能够对多达 20 个 miRNA 进行多重检测，而单分子分析平台将实现无 PCR 检测，灵敏度 <20 个拷贝，样品体积 <10 pL。这种高灵敏度和接近零样本量的独特组合将为 II 期单细胞 miRNA 分析平台奠定基础。此外，该架构可以通过微流体轻松扩展到更高程度的复用（> 50 重）和吞吐量。在目标 1 中，我们将使用合成 RNA 靶点来开发基本的 Ligo-miR 测定法，以模拟 3 种经典 miRNA：let-7a、miR-16 和 miR-21。在目标 2 中，我们将设计一个 SML-FSHS 微流体装置来分析目标 1 中生成的连接产物。在目标 3 中，我们将把这些技术集成到多重检测中，每个反应可以检测 20 个 miRNA。最后，在目标 4 中，我们将使用 20 重检测来分析 3 个人类癌细胞系和 3 个正常组织中的 miRNA。 Ligo-miR 结果将与已发表的微阵列和 RT-PCR 数据进行比较。这种方法不仅适用于 miRNA 肿瘤分析，还适用于稀有样本的其他应用，例如使用循环肿瘤细胞和无细胞 miRNA。