HIGH DENSITY MICROARRAYS WITH QUANTITATIVE QC/QA

具有定量 QC/QA 功能的高密度微阵列

• 批准号: 6781772
• 负责人: Martin J Hessner
• 金额: $ 37.5万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6781772
• 关键词: biotechnology; computer data analysis; computer program /software; cost effectiveness; data collection methodology /evaluation; data management; data quality /integrity; fluorescent dye /probe; gene expression; image processing; laboratory rat; mathematical model; microarray technology; molecular cloning; nucleic acid hybridization; nucleic acid probes; polymerase chain reaction; technology /technique development

DESCRIPTION (provided by applicant): DNA microarray analysis has become a widely used genetic tool; however, its massive scale creates quality control and analysis problems for most laboratories wishing to use it. The purpose of this proposal is to facilitate the application of this technology to fundamental hypothesis-driven research through the identification and resolution of the sources of cDNA microarray data variability. Since the array itself is a considerable source of variability we have developed a three-color array platform that allows prehybridization array visualization. Our direct labeling approach separates assessment of slide coating, printing and blocking from hybridization, enabling evaluation of element/array morphology, surface DNA deposition/retention, and background levels prior to hybridization. We have also developed Matarray, a software package for hybridized image processing and data acquisition that provides quantitative QC for each array element as well as the entire slide. Combined, these efforts provide the basis for an integrated quality control (QC) approach to effectively manage microarray technical and systematic variability. For this application we will build upon these initial efforts through the following specific aims 1) We will study the relationship between measurable prehybridization image quality parameters and data quality/reproducibility, including the influence of background, spot morphology, and bound probe. Data compression caused by limiting bound probe will be quantified using the third dye so that data derived from sub-optimal spots can either be normalized or filtered. 2) We will develop pre-hybridization array qualification software based upon parameters identified in aim 1, which will encompass probe plate tracking, quantification of probe available for hybridization, and array/element morphology. Post-hybridization algorithms will be developed that utilize third dye information for data normalization and filtering. 3) Our improved microarray and analysis platforms will be validated by expression profiling disease development in an animal system. The commercial oligonucleotide and cDNA arrays will be used as reference platforms to assess cost effectiveness and value of our approach. Our success will provide an accurate, flexible, lower cost alternative to commercial arrays for laboratories wishing to conduct gene expression studies.

描述（申请人提供）：DNA微阵列分析已成为广泛使用的遗传工具；然而，它的庞大规模给大多数希望使用它的实验室带来了质量控制和分析问题。本提案的目的是通过鉴定和解决cDNA微阵列数据变异性的来源，促进该技术在基础假设驱动研究中的应用。由于阵列本身是一个相当大的可变性来源，我们已经开发了一个三色阵列平台，允许预杂交阵列可视化。我们的直接标记方法将载玻片涂层、打印和阻断的评估与杂交分离开来，从而能够评估元件/阵列形态、表面DNA沉积/保留以及杂交前的背景水平。我们还开发了Matarray，这是一个用于杂交图像处理和数据采集的软件包，可为每个阵列元素以及整个幻灯片提供定量QC。结合起来，这些努力为有效管理微阵列技术和系统可变性的综合质量控制（QC）方法提供了基础。对于本应用程序，我们将通过以下具体目标建立在这些初步努力的基础上：1)我们将研究可测量的预杂交图像质量参数与数据质量/再现性之间的关系，包括背景，斑点形态和结合探针的影响。将使用第三种染料对限定界探针引起的数据压缩进行量化，以便从次优点导出的数据可以归一化或过滤。2)我们将根据目标1中确定的参数开发预杂交阵列鉴定软件，该软件将包括探针板跟踪，可用于杂交的探针定量以及阵列/元件形态。后杂交算法将开发利用第三染料信息的数据归一化和滤波。3)我们改进的微阵列和分析平台将在动物系统中通过表达谱分析疾病发展进行验证。商业寡核苷酸和cDNA阵列将作为参考平台来评估我们的方法的成本效益和价值。我们的成功将为希望进行基因表达研究的实验室提供一种准确、灵活、低成本的商业阵列替代方案。