HIGH DENSITY MICROARRAYS WITH QUANTITATIVE QC/QA

描述（申请人提供）：DNA微阵列分析已成为一种广泛使用的遗传学工具;然而，它的大规模产生的质量控制和分析问题，希望使用它的大多数实验室。本提案的目的是通过识别和解决cDNA微阵列数据变异性的来源，促进该技术在基本假设驱动的研究中的应用。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布、印刷和阻断的评估与杂交分开，从而能够在杂交之前评估元件/阵列形态、表面DNA沉积/保留和背景水平。我们还开发了Matarray，这是一种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载玻片提供定量QC。结合起来，这些努力提供了一个综合的质量控制（QC）的方法，以有效地管理微阵列技术和系统的变异性的基础。对于该应用，我们将通过以下具体目标建立在这些初始努力的基础上1）我们将研究可测量的预杂交图像质量参数和数据质量/再现性之间的关系，包括背景、斑点形态和结合探针的影响。将使用第三种染料定量由限制结合探针引起的数据压缩，使得可以归一化或过滤源自次优斑点的数据。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，其中包括探针板跟踪、可用于杂交的探针定量和阵列/元件形态。将开发利用第三染料信息进行数据归一化和过滤的杂交后算法。3)我们改进的微阵列和分析平台将通过动物系统中的表达谱疾病发展来验证。商业寡核苷酸和cDNA阵列将被用作参考平台，以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准确、灵活、低成本的商业阵列替代品。

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.