Enzymatic Tools for Degrading Tissue and Preserving RNA

用于降解组织和保存 RNA 的酶工具

• 批准号: 6548165
• 负责人: GARY J LATHAM
• 金额: $ 16.24万
• 依托单位: AMBION, INC.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-27 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6548165
• 关键词: collagenase; endopeptidases; enzyme activity; enzyme complex; enzyme mechanism; gene expression; high throughput technology; lipase; nuclease; nucleic acid purification; nucleic acid quantitation /detection; technology /technique development; tissue /cell culture

DESCRIPTION (provided by applicant): Intact RNA is an obligate starting material for expression profiling technologies that can link specific molecular events with disease phenotypes. RNA isolations from tissue currently require invasive mechanical force to disrupt the cellular architecture. Such mechanical approaches are tedious and potentially inefficient. They also expose the operator to possible biohazards. As a result, the development of a "closed system" for RNA isolation is needed to enable the high-throughput recovery of RNA from tissues such as biopsies using a safeguarded platform that can process dozens or hundreds of tissues at once. We proposed to achieve this goal through the use of "MELT" (Multi-Enzymatic Liquefaction of Tissue) technology. This technology will harness the degrading power of enzymes that have evolved together as a biological tool set for the decomposition of complex food sources. We refer to this tool set as an "enzyme community." Since some enzyme communities (e.g., the secreted enzymes of bacterial pathogens) mimic the spectrum of activities needed to digest tissue, these collections are an ideal repository for tissue-degrading enzymes. We propose to characterize and blend enzyme activities to create a MELT reagent that can rapidly degrade tissues, "freeze" mRNA expression profiles, and stabilize the RNA for downstream analyses.

描述（由申请人提供）： 完整RNA是表达谱分析的专性起始材料 可以将特定分子事件与疾病表型联系起来的技术。 目前，从组织中分离RNA需要侵入性机械力， 破坏细胞结构。这种机械方法是乏味的， 潜在的低效率。它们还使操作员暴露于可能的生物危害。 因此，需要开发用于RNA分离的"封闭系统 为了能够从组织如活组织检查中高通量地回收RNA 使用一个安全的平台，可以处理几十个或几百个组织， 一次我们建议通过使用“MELT”来实现这一目标 （组织的多酶液化）技术。这项技术将 利用酶的降解能力， 分解复杂食物来源的生物工具集。我们称 这个工具集是一个"酶社区"。"由于一些酶群落（例如， 细菌病原体的分泌酶）模拟活性谱 需要消化组织，这些集合是一个理想的储存库， 组织降解酶我们建议表征和混合酶 活动，以创造一种MELT试剂，可以迅速降解组织，"冻结" mRNA表达谱，并稳定RNA用于下游分析。