Novel Method for Micro RNA Amplification

描述（由申请人提供）：项目摘要/摘要：细胞microRNA（miRNAs）是一类新发现的单链RNA分子，在植物和动物细胞中表达。估计有500-700个miRNA基因，到目前为止，每个物种已经鉴定了大约260个。miRNAS已被证明是通过翻译抑制和mRNA降解的基因表达的负调节剂。每个miRNAs的精确功能是目前大量研究工作的主题。miRNA的表达是高度调节和协调的，不同的细胞类型在不同的生理条件下似乎有独特的miRNA表达模式。越来越多的证据表明，miRNA的表达模式不仅可以指示细胞或组织的生理状态，还可以指示疾病状态，例如癌症。需要高灵敏度的工具来检测和表达miRNA谱。北方印迹是检测和定量miRNA的金标准。北方印迹是一个相对繁琐的过程，灵敏度低。需要用于高通量和灵敏度的方法。成熟的miRNA长度为17-24个核苷酸;由于它们的小尺寸和单链性质，难以通过常规聚合酶链式反应（PCR）实现有效和忠实的扩增。大多数目前的检测和分析方法依赖于信号的放大;这些方法的灵敏度仍然远远低于PCR扩增所能达到的水平。在本项目中，我们提出了一种新的基于多重PCR的miRNA扩增系统。扩增系统被设计成忠实地同时扩增所有miRNA而没有偏差。该扩增系统的商业应用很多，例如用于通过实时PCR定量miRNA，用于诊断目的所需的高灵敏度检测，以及用于miRNA表达谱分析。第一阶段的研究将验证这一过程;第二阶段将进一步开发新的miRNA扩增系统的商业应用。项目简介：本项目旨在开发一种用于研究和诊断目的的新技术。所开发的技术将为生物医学研究和疾病诊断提供必要的工具， 例如癌症，这肯定会对整个公共卫生产生重大影响。

DESCRIPTION (provided by applicant): Project Summary/Abstract: Cellular microRNAs (miRNAs) are a newly discovered class of single-stranded RNA molecules that are expressed in cells of plants and animals. There are an estimated 500-700 miRNA genes for which about 260 have been identified for each species so far. miRNAS have been demonstrated to be negative regulators of gene-expression via translation inhibition and mRNA degradation. Precise function for each miRNAs is currently the subject of intense research effort. miRNA expression is highly regulated and coordinated, different cell types under different physiological conditions appear to have a unique miRNA expression pattern. Increasing evidence suggests miRNA expression pattern could be indicative not only of the physiological state but also the disease state of cell or tissue such as cancer. Highly sensitive tools are needed for detection and expression profiling of miRNAs. Northern blot is the gold-standard for detection and quantification of miRNAs. Northern blot is a comparatively tedious process with low sensitivity. Methods for high throughput and sensitivity are needed. Mature miRNAs are from 17-24 nucleotides long; because their mall size and single stranded nature, efficient and faithful amplification by conventional polymerase chain reaction (PCR) is difficult to achieve. Most current methods for detection and profiling rely on the amplification of signals; the sensitivity of those methods is still far below the level can be achieved with PCR amplification. In this project, we propose to develop a novel multiplex PCR based miRNA amplification system. The amplification system is designed to faithfully amplify all miRNAs simultaneously without bias. Commercial applications for this amplification system are numerous, such as for quantitation of miRNA by real-time PCR, for highly sensitive detection required for diagnostic purpose, and for miRNA expression profiling. The Phase I research will validate the process; Phase II will further develop commercial applications for the novel miRNA amplification system. Project Narrative: This project is to develop a novel technology for the research and diagnostic purposes. The developed technology will provide essential tools for biomedical research and for disease diagnostics, such as cancer, for which will definitely have a great impact in public health in general.