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

描述（由申请人提供）：用于miRNA分析的Ligo-miR - A多重单分子连接试验MicroRNA（miRNA）是短的非编码RNA，在细胞过程（如分化）和疾病状态（如癌症）的整个基因表达中具有普遍作用。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的关键步骤。在肿瘤块中以单细胞分辨率分析miRNA表达的能力可能导致有效消除而不仅仅是缩小肿瘤的靶向治疗。现有的miRNA分析方法没有将高灵敏度与真正的多路复用和小体积能力相结合。在这个I期研究计划中，将开发一种用于miRNA谱分析的无PCR、多重连接测定法，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。然后使用微流体无单分子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能够在每个反应中进行多达20个miRNA的多重检测，而单分子分析平台将使无PCR检测具有<20个拷贝的灵敏度和<10 pL的样品体积。这种高灵敏度和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，这种架构可以通过微流体容易地扩展到甚至更高程度的多路复用（>50-plex）和吞吐量。在目标1中，我们将开发基本的Ligo-miR检测，使用合成的RNA靶标模拟3种经典的miRNA，let-7a，miR-16和miR-21。在目标2中，我们将设计一个SML-FSHS微流控装置来分析目标1中产生的连接产物。在目标3中，我们将这些技术整合到一个多重检测中，每个反应可以检测20个miRNA。最后，在目标4中，我们将使用20-plex测定来分析3种人类癌细胞系和3种正常组织中的miRNA。然后将Ligo-miR结果与已发表的微阵列和RT-PCR数据进行比较。这种方法不仅在miRNA肿瘤谱分析中具有应用，而且在具有稀有样品的其他应用中具有应用，例如使用miRNA的临床诊断。 循环肿瘤细胞和无细胞miRNA。

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.