Novel Method for Micro RNA Amplification

Micro RNA 扩增新方法

• 批准号: 7161230
• 负责人: Donald Rao
• 金额: $ 10.73万
• 依托单位: SILERETECH, LLC
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-15 至 2007-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7161230
• 关键词: biotechnology; gene expression profiling; high throughput technology; method development; microRNAs; microarray technology; nucleic acid amplification techniques; nucleic acid chemical synthesis; nucleic acid quantitation /detection; polymerase chain reaction

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.

描述（由申请人提供）： 项目摘要/摘要：细胞 microRNA (miRNA) 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，迄今为止每个物种已鉴定出约 260 个基因。 miRNAS 已被证明通过翻译抑制和 mRNA 降解而成为基因表达的负调节因子。每个 miRNA 的精确功能是目前深入研究的主题。 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似乎具有独特的miRNA表达模式。越来越多的证据表明，miRNA 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，例如癌症。 miRNA 的检测和表达谱分析需要高度灵敏的工具。 Northern blot 是 miRNA 检测和定量的金标准。 Northern blot是一个相对繁琐的过程，灵敏度较低。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；由于它们尺寸小且单链性质，传统聚合酶链式反应 (PCR) 很难实现高效、忠实的扩增。目前大多数检测和分析方法都依赖于信号放大。这些方法的灵敏度仍远低于PCR扩增所能达到的水平。在这个项目中，我们建议开发一种新型的基于多重PCR的miRNA扩增系统。扩增系统旨在无偏差地同时忠实地扩增所有 miRNA。该扩增系统的商业应用有很多，例如通过实时 PCR 定量 miRNA、诊断目的所需的高灵敏度检测以及 miRNA 表达谱分析。第一阶段研究将验证该过程； II 期将进一步开发新型 miRNA 扩增系统的商业应用。项目叙述：该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健康产生巨大影响。