ASSAY FOR RNA INTEGRITY AND OPTIMIZATION OF RNA RECOVERY

需要改进的方法来保存、分离和鉴定信使核糖核酸。现有的RNA提取技术足以用于低分辨率分析，如Northern杂交。然而，构建高质量的cDNA文库和利用高分辨率技术分析差异基因表达都对样品RNA完整性的微小变化敏感。通常，这些差异是由次优处理引起的，并可以通过优化RNA分离来最小化。然而，RNA质量的变化有时是被分析样本固有的。例如，肿瘤活检组织中的坏死细胞含有降解的RNA。在任何一种情况下，在使用劳动密集型技术进行高分辨率分析之前识别这样的变异是至关重要的。我们建议建立一种RNA完整性的定量检测方法，用于在用高分辨率技术构建或分析cDNA文库之前对RNA样品进行资格预审。我们还希望制定指南，将RNA的完整性与这些技术中使用的适宜性联系起来。这些信息将是学术和工业实验室的宝贵资源。最后，我们建议利用这一方法对RNA提取技术进行系统的优化，以最大化回收的RNA的完整性。拟议的商业应用：Ambion是一家领先的RNA定量和分析产品供应商，该公司打算销售一种RNA完整性的定量检测、“认证质量”的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.