Ligo-miR - A Multiplex Assay Platform for Targeted miRNA Profiling

 描述（由申请人提供）：miRNA具有彻底改变癌症检测和治疗的潜力，因为它们几乎控制从分化到生长和衰老的每一个细胞过程。尽管预计存在超过2000种miRNA，但研究表明，仅5-50种miRNA的较小子集通常是任何特定疾病或细胞过程中的关键参与者。然而，现有的微阵列和qRT-PCR技术太慢且太昂贵，无法在这些靶向miRNA组中筛选大量样本，而这是验证miRNA癌症测试的关键任务。在第一阶段，我们开发了一种多重连接测定法，在称为Ligo-miR的单一反应中分析10-100种miRNA。我们证明Ligo-miR表达谱与金标准Taqman qRT-PCR测定具有高度相关性（R2=0.89-98）。然而，Ligo-miR所需的动手时间减少了10- 20倍，成本降低了5倍，总体样品通量提高了20倍，成功实现了快速，低成本，靶向miRNA分析的目标。Ligo-miR结合了2个连续的连接步骤，以产生由长度编码的miRNA特异性反应产物。这种灵活的、基于长度的多路复用被专门设计成能够使用一系列DNA大小测定方法（包括聚丙烯酰胺凝胶电泳（PAGE）、毛细管电泳（CE）和单分子自由溶液流体动力学分离（SML-FSHS））进行检测，使得灵敏度、多路复用能力和样品通量可以针对广泛的miRNA应用和用户需求进行定制。在第二阶段SBIR提案中，我们将利用第一阶段的结果开发一个基于Ligo-miR技术的商业产品平台，用于广泛的研究应用。首先，Ligo-miR EZ将被开发用于细胞和组织的一般分析，使用 荧光PAGE检测，以实现每个细胞20个拷贝的灵敏度。几乎所有的分子实验室都将拥有热循环仪和PAGE设备，使所有人都可以进行快速和低成本的多重分析。接下来，Ligo-miR CE将被开发用于需要最高灵敏度和通量的应用，如单细胞分析或血液临床分析。它结合了自动化CE检测，以增加样品吞吐量，每天数千个样品和后扩增，以提高灵敏度，单细胞水平。然后，Ligo-miR HD将 开发用于具有挑战性的应用，使用有限的样品或可以受益于芯片实验室集成，如FFPE分析和单细胞。它使用微流控单分子技术，使用微量样品进行高灵敏度检测。最后，我们将对每个Ligo-miR检测试剂盒进行技术验证，并以Taqman qRT-PCR为基准。

 DESCRIPTION (provided by applicant): MiRNA have the potential to revolutionize both cancer detection and therapeutics as they exert control over nearly every cellular process from differentiation to growth and senescence. Although over 2000 miRNA are predicted to exist, studies have suggested that smaller subsets of only 5-50 miRNA are often the critical players in any specific disease or cellular process. However, existing microarray and qRT-PCR techniques are too slow and too expensive to screen large numbers of samples across these targeted miRNA panels, a critical task in the validation of miRNA cancer tests. In Phase I, we developed a multiplexed ligation assay to analyze 10-100 miRNA in a single reaction called Ligo-miR. We demonstrated that Ligo-miR expression profiles had high correlation to the gold standard Taqman qRT-PCR assays (R2=0.89-98). Yet, Ligo-miR required 10-20x less hands-on time, had 5x less cost, and had 20x higher overall sample-throughput, successfully achieving the goal of fast and low-cost, targeted miRNA profiling. Ligo-miR incorporates 2 sequential ligation steps to generate miRNA specific reaction products encoded by length. This flexible, length-based multiplexing is specifically designed to enable detection using an array of DNA sizing methods including polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), and single molecule free solution hydrodynamic separation (SML-FSHS) such that sensitivity, multiplex capability, and sample throughput can be tailored a wide spectrum of miRNA applications and user requirements. In this Phase II SBIR proposal, we will leverage the Phase I results to develop a platform of commercial products based on Ligo-miR technology for broad research applications. First, Ligo-miR EZ will be developed for general profiling of cells and tissues using fluorescent PAGE detection to achieve a sensitivity of 20 copies per cell. Nearly all molecular labs will have a thermal cycler and PAGE apparatus, making fast and low cost multiplex analysis available to all. Next, Ligo- miR CE will be developed for applications requiring the highest sensitivity and throughput such as single cell analysis or clinical analysis of blood. It incorporates automated CE detection to increase sample throughput to thousands of samples per day and a post-amplification to boost sensitivity to single cell levels. Then, Ligo-miR HD will be developed for challenging applications using limited samples or that could benefit from lab-on-a-chip integration such as FFPE analysis and single cell. It uses microfluidic, single molecule technology for high sensitivity detection using minute amounts of sample. Finally, we will perform a technical validation and benchmark each Ligo-miR assay against Taqman qRT-PCR.