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

描述(由申请人提供)：LIGO-miR-用于miRNA分析的多路复用单分子连接分析microRNAs(MiRNA)是一种短的、非编码的RNA，在分化和癌症等疾病状态等细胞过程中普遍发挥着基因表达的作用。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的疾病治疗的关键步骤。能够在单个细胞分辨率下对肿瘤块进行miRNA表达的能力可能会导致靶向治疗，这种治疗在消除肿瘤方面有效，而不仅仅是缩小肿瘤。现有的miRNA分析方法没有一种将高灵敏度与真正的多路复用和小体积能力相结合。在这个第一阶段转换方案中，将开发一种称为LIGO-miR的不含聚合酶链式反应的多重连接试验，用于miRNA图谱分析。锁定的核酸探针的杂交和连接将被用来产生按长度编码的miRNA特异连接产物。然后，将使用微流体单分子无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能够对每个反应进行多达20个miRNA的多重检测，而单分子分析平台将实现无聚合酶链式反应检测，灵敏度为&lt；20个拷贝，样本体积&lt；10pl。这种高灵敏度和接近于零的样本量的独特组合将为第二阶段单细胞miRNA分析平台奠定基础。此外，这种架构可以很容易地扩展到更高程度的多路复用(&gt；50-plex)和通过微流体的吞吐量。在目标1中，我们将使用合成的RNA靶来模拟3个经典的miRNAs，let-7a，miR-16和miR-21，建立基本的Ligo-miR分析。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，我们将把这些技术集成到一个多重分析中，每次反应可以检测到20个miRNAs。最后，在目标4中，我们将使用20-plex方法来分析3个人类癌细胞株和3个正常组织中的miRNA。然后将LIGO-miR结果与公布的微阵列和RT-PCR数据进行比较。这种方法不仅在miRNA肿瘤分析中有应用，而且在其他稀有样本的应用中也有应用，例如使用 循环肿瘤细胞和无细胞miRNA。 与公共健康相关：LIGO-miR-一种多重单分子连接分析miRNA图谱microRNAs最近被发现在基因表达调控中发挥关键作用。因此，开发一种灵敏的、新的microRNA分析平台将有助于更好地了解肿瘤的发展和癌症的进展。反过来，这可能导致更特异和更强大的癌症生物标记物和更有效的、有针对性的癌症治疗。

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. PUBLIC HEALTH RELEVANCE: Ligo-miR - A Multiplexed Single Molecule Ligation Assay for miRNA profiling MicroRNAs have been recently found to play key roles throughout the regulation of gene expression. Thus, the development of a sensitive, new assay platform for microRNA analysis will lead to better understanding of tumor development and cancer progression. In turn, this could lead more specific and robust cancer biomarkers and more effective, targeted cancer therapies.