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）的基因表达中具有普遍作用。揭示这些分子在发展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。通过肿瘤块在单细胞分辨率上分析miRNA表达的能力可能会导致有效消除而不仅仅是缩小肿瘤的靶向治疗。没有一种现有的miRNA分析方法能兼具高灵敏度、真正的复用性和小体积能力。在这个I期SHIFT提案中，将开发一种无pcr的，用于miRNA分析的多重连接试验，称为Ligo-miR。将锁定的核酸探针进行杂交和连接，生成以长度编码的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）直接鉴定和定量结扎产物。这种连接机制将使Ligo-miR能够在每次反应中进行多达20个miRNA的多重检测，而单分子分析平台将实现无pcr检测，灵敏度<20拷贝，样样量<10 pL。这种高灵敏度和接近于零的样样量的独特组合将为II期单细胞miRNA分析平台奠定基础。此外，这种架构可以很容易地扩展到更高程度的多路复用（bbb50 -plex）和通过微流体的吞吐量。在Aim 1中，我们将开发基本的Ligo-miR分析，使用合成RNA靶标来模拟3种经典的mirna， let-7a， miR-16和miR-21。在Aim 2中，我们将设计一个SML-FSHS微流控装置来分析Aim 1中产生的结扎产物。在Aim 3中，我们将把这些技术整合到一个多重分析中，每个反应可以检测20个mirna。最后，在Aim 4中，我们将使用20-plex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。该方法不仅在miRNA肿瘤分析中有应用，而且在临床诊断等罕见样本应用中也有应用

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.