Rapid Quantitation of Small RNA

描述(申请人提供)：microRNAs(MiRNAs)是一类重要的新生物分子，具有促进人类健康的潜力。在过去的三年里，随着研究人员发现这些小RNA在人类癌症、神经疾病和病毒感染中的潜在重要性，miRNA的出版物呈指数级增长。Ambion的研究人员致力于开发必要的工具，以了解miRNAs与人类健康之间的关系。我们的结果和其他机构的结果表明，miRNAs可能为诊断和预后分析的发展提供有用的标记物。此外，有迹象表明，miRNAs可能在疾病进展中发挥积极作用，使它们成为治疗干预的对象。这项提议的目标是开发快速、灵敏、高通量的方法来量化生物样品中微小RNA(MiRNA)等小RNA分子的水平。这些小RNA已经成为基因表达的重要调节者，而基因表达对发育和疾病进展至关重要。不幸的是，目前还没有快速、定量的方法来测量临床样本中的miRNA水平。这项拟议的研究将导致开发试剂盒和试剂，以快速定量来自组织或细胞培养的样本中的miRNAs。在第一阶段，我们发明了一种基于qRT-PCR的方法，将目标miRNA分子转化为cDNA，然后进行扩增和PCR检测。该方法能够特异性地检测RNA样品中的单个miRNAs。在第二阶段，我们建议优化该方法，以促进其在研究和诊断应用中的商业应用。我们的第二阶段研究将提高灵敏度和特异度，允许在单一反应中同时检测多个miRNAs，并允许在Luminex液体珠阵列平台上进行检测。这些改进将使基础研究的快速miRNA分析、诊断和治疗miRNA候选的临床验证成为可能，并为快速开发基于miRNA的临床诊断分析建立一个系统。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are a significant new class of biomolecules with potential to enhance human health. In the last three years, miRNA publications have increased exponentially as researchers have discovered the potential importance of these small RNAs in human cancer, neurological disease, and viral infection. Ambion researchers have worked to develop the tools necessary to understand the relationship between miRNAs and human health. Our results and the results from other institutions indicate that miRNAs may provide useful markers for the development of diagnostic and prognostic assays. In addition, there are hints that miRNAs might play an active role in disease progression, making them candidates for therapeutic intervention. The goal of this proposal is to develop rapid, sensitive, high-throughput methods to quantify the levels of small RNA molecules such as microRNAs (miRNA) in biological samples. These small RNAs have emerged as important regulators of gene expression which are critical for development and disease progression. Unfortunately, there currently are no rapid, quantitative methods available to measure levels of miRNA in clinical samples. The proposed research will result in the development of kits and reagents to rapidly quantify miRNAs in samples derived from tissues or cell culture. During Phase I, we invented a qRT-PCR based method to convert target miRNA molecules into cDNA and which are then amplified and detected using PCR. This method enables the specific detection of individual miRNAs in RNA samples. In Phase II we propose to optimize the method to facilitate its commercial use in research and diagnostic applications. Our Phase II research will improve sensitivity and specificity, permit simultaneous detection of multiple miRNAs in a single reaction, and enable detection on a Luminex liquid bead array platform. These improvements will enable rapid miRNA profiling for basic research, clinical validation of diagnostic and therapeutic miRNA candidates, and put a system in place for rapid development of miRNA-based clinical diagnostic assays.