Rapid Quantitation of Small RNA

描述（由申请人提供）：微小RNA（miRNA）是一类重要的新型生物分子，具有增强人类健康的潜力。在过去的三年里，miRNA的出版物呈指数级增长，因为研究人员发现了这些小RNA在人类癌症，神经系统疾病和病毒感染中的潜在重要性。Ambion的研究人员一直致力于开发必要的工具来了解miRNAs与人类健康之间的关系。我们的研究结果和其他机构的研究结果表明，miRNAs可能为诊断和预后检测的发展提供有用的标志物。此外，有迹象表明miRNAs可能在疾病进展中发挥积极作用，使其成为治疗干预的候选者。该提案的目标是开发快速，灵敏，高通量的方法来定量生物样品中小RNA分子如microRNA（miRNA）的水平。这些小RNA已成为基因表达的重要调节因子，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miRNA的水平。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物的样品中的miRNA。在第一阶段，我们发明了一种基于qRT-PCR的方法，将靶miRNA分子转化为cDNA，然后使用PCR扩增和检测。该方法能够特异性检测RNA样品中的单个miRNA。在第二阶段，我们建议优化方法，以促进其在研究和诊断应用中的商业用途。我们的II期研究将提高灵敏度和特异性，允许在单个反应中同时检测多种miRNA，并实现在Luminex液体微珠阵列平台上的检测。这些改进将使基础研究、诊断和治疗候选miRNA的临床验证能够快速进行miRNA谱分析，并为快速开发基于miRNA的临床诊断检测提供一个系统。

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.