ASSAY FOR RNA INTEGRITY AND OPTIMIZATION OF RNA RECOVERY

需要改进mRNA的保存、分离和表征方法。现有的RNA分离技术足以用于低分辨率分析，例如北方杂交。然而，构建高质量的cDNA文库和使用高分辨率技术分析差异基因表达对样品RNA完整性的微小变化都很敏感。通常，这些变化是由次优处理引起的，可以通过优化RNA分离来最小化。然而，RNA质量的变化有时是被分析样品固有的。例如，肿瘤活检中的坏死细胞含有降解的RNA。在任何一种情况下，都必须在使用劳动密集型技术进行高分辨率分析之前确定这些变化。我们建议开发一种用于RNA完整性的定量测定方法，用于在cDNA文库构建或高分辨率技术分析之前对RNA样品进行预鉴定。我们还希望制定指导方针，将RNA的完整性与这些技术的适用性联系起来。这些信息将是学术和工业实验室的宝贵资源。最后，我们建议使用这种方法进行RNA分离技术的系统优化，目的是最大限度地提高回收RNA的完整性。拟议的商业应用：Ambion是RNA定量和分析产品的领先供应商，打算推出一种RNA完整性定量检测、“质量认证”RNA和改进的RNA分离系统。

Improved methodologies for the preservation, isolation, and characterization of mRNA are needed. Existing RNA isolation technologies are sufficient for low-resolution analysis such as northern hybridization. However, construction of quality cDNA libraries and the analysis of differential gene expression using high-resolution techniques are both sensitive to small variations in the integrity of sample RNA. Frequently, these variations are caused by sub-optimal handling and can be minimized by optimizing RNA isolation. However, variations in RNA quality are sometimes intrinsic to the samples being analyzed. For example, necrotic cells within tumor biopsies contain degraded RNA. In either case, it is essential that such variations be identified prior to the expense of high-resolution analysis using labor intensive techniques. We propose to develop a quantitative assay for RNA integrity to be used to pre-qualify RNA samples prior to use in cDNA library construction or analysis by high resolution techniques. We also wish to develop guidelines to relate intactness of RNA to suitability for use in these techniques. Such information will be an invaluable resource for both academic and industrial laboratories. Finally, we propose to use this assay for a systematic optimization of RNA isolation technologies, with the aim of maximizing the intactness of recovered RNA. PROPOSED COMMERCIAL APPLICATIONS: Ambion, a leading supplier of products for RNA quantitation and analysis, intends to market a quantitative assay for RNA integrity, 'Certified Quality' RNA, and improved RNA isolation systems.