Novel Method for Micro RNA Amplification

项目概述/摘要：细胞微RNA （Cellular microRNAs, 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，每个物种已鉴定出约260个。miRNAS通过翻译抑制和mRNA降解被证明是基因表达的负调控因子。每种mirna的精确功能是目前激烈研究的主题。miRNA的表达是高度调控和协调的，不同的细胞类型在不同的生理条件下表现出独特的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生理状态，还可以指示细胞或组织的疾病状态，如癌症。需要高灵敏度的工具来检测和分析mirna的表达。Northern blot是检测和定量mirna的金标准。北方印迹是一个相对乏味的过程，灵敏度低。需要高通量和高灵敏度的方法。成熟的mirna长度为17-24个核苷酸；由于其体积小且单链的性质，传统的聚合酶链反应（PCR）难以实现高效和忠实的扩增。目前大多数检测和分析方法依赖于信号的放大；这些方法的灵敏度仍远低于PCR扩增所能达到的水平。在本项目中，我们提出开发一种新的基于多重PCR的miRNA扩增系统。该扩增系统旨在忠实地同时扩增所有mirna而不存在偏见。该扩增系统的商业应用非常广泛，例如通过实时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型miRNA扩增系统的商业应用。项目描述：本项目旨在开发一种用于研究和诊断目的的新技术。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，

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.