Identification and removal of contaminating fluorescence from commercial and in-house printed DNA microarrays
Identification and removal of contaminating fluorescence from commercial and in-house printed DNA microarrays
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DOI：
10.1093/nar/gng018
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发表时间：
2003-02-15
影响因子：
14.9
通讯作者：
Werner-Washburne, M
中科院分区：
文献类型：
--
作者：
Martinez, MJ;Aragon, AD;Werner-Washburne, M
微阵列分析是基因组生物学的一项至关重要的技术，因此获得的数据可靠至关重要。目前用于微阵列分析的软件和标准化技术依赖于这样的假设，即斑点内的荧光背景在整个载玻片上基本上是相同的，并且可以通过斑点周围的荧光来测量。如果背景荧光是斑点定位的，则该假设是无效的。斑点下背景荧光的不准确估计产生误差源，特别是对于低表达基因。我们已经在几个商业和内部打印的微阵列载玻片上的Cy3通道中鉴定出斑点定位的污染荧光。我们通过模拟杂交（没有标记的靶标）确定，预杂交扫描不能用于预测杂交后这种污染荧光的贡献，因为杂交后斑点间荧光的变化太可变。确定了解决这一问题的两个办法。首先，在打印到Corning UltraGAPS载玻片上之前，允许4小时暴露于空气中，将污染荧光强度显著降低到接近周围玻璃的值。或者，应用一种新的，高光谱成像扫描仪和多元曲线分辨率算法，允许光谱贡献的Cy3信号，玻璃，和污染的荧光杂交后进行区分和定量。
Microarray analysis is a critically important technology for genome-enabled biology, therefore it is essential that the data obtained be reliable. Current software and normalization techniques for microarray analysis rely on the assumption that fluorescent background within spots is essentially the same throughout the glass slide and can be measured by fluorescence surrounding the spots. This assumption is not valid if background fluorescence is spot-localized. Inaccurate estimates of background fluorescence under the spot create a source of error, especially for low expressed genes. We have identified spot-localized, contaminating fluorescence in the Cy3 channel on several commercial and in-house printed microarray slides. We determined through mock hybridizations (without labeled target) that pre-hybridization scans could not be used to predict the contribution of this contaminating fluorescence after hybridization because the change in spot-to-spot fluorescence after hybridization was too variable. Two solutions to this problem were identified. First, allowing 4 h of exposure to air prior to printing on to Corning UltraGAPS slides significantly reduced contaminating fluorescence intensities to approximately the value of the surrounding glass. Alternatively, application of a novel, hyperspectral imaging scanner and multivariate curve resolution algorithms, allowed the spectral contributions of Cy3 signal, glass, and contaminating fluorescence to be distinguished and quantified after hybridization.