Label-Free Prehybridization DNA Microarray Imaging Using Photonic Crystals for Quantitative Spot Quality Analysis
Label-Free Prehybridization DNA Microarray Imaging Using Photonic Crystals for Quantitative Spot Quality Analysis
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DOI：
10.1021/ac101551c
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发表时间：
2010-10-15
影响因子：
7.4
通讯作者：
Cunningham, B. T.
中科院分区：
文献类型：
--
作者：
George, S.;Block, I. D.;Cunningham, B. T.
DNA捕获探针打印过程中的技术变异性仍然是从微阵列实验中获得高质量数据的重要障碍。虽然已经提出了在杂交前使用荧光标记对印刷阵列进行可视化的方法，但使用无标记技术测量斑点密度的能力将提供关于斑点质量的有价值的信息，而不会改变标准的微阵列方案。在这项研究中，我们介绍了使用光子晶体生物传感器表面和高分辨率无标记成像检测仪器来产生斑点寡核苷酸微阵列的预杂交图像。从这些图像测量光斑强度、大小、饱和程度和局部背景强度。这些信息被用于自动识别遗漏的斑点(由于机械故障或样品耗尽)，以及对反映每个印刷特征的质量的分数进行分配。漏掉的斑点以95%的敏感度识别。此外，基于斑点质量分数的过滤增加了复制阵列之间杂交后斑点强度的成对相关性，表明标签树斑点质量分数捕捉到了微阵列数据中的变异性。该成像方法可用于印制的基因芯片、寡核苷酸芯片和蛋白质芯片的质量控制。
Technical variability during DNA capture probe printing remains an important obstacle to obtaining high quality data from microarray experiments. While methods that use fluorescent labels for visualizing printed arrays prior to hybridization have been presented, the ability to measure spot density using label-free techniques would provide valuable information on spot quality without altering standard microarray protocols. In this study, we present the use of a photonic crystal biosensor surface and a high resolution label-free imaging detection instrument to generate prehybridization images of spotted oligonucleotide microarrays. Spot intensity, size, level of saturation, and local background intensity were measured from these images. This information was used for the automated identification of missed spots (due to mechanical failure or sample depletion) as well as the assignment of a score that reflected the quality of each printed feature. Missed spots were identified with >95% sensitivity. Furthermore, filtering based on spot quality scores increased pairwise correlation of posthybridization spot intensity between replicate arrays, demonstrating that label-tree spot quality scores captured the variability in the microarray data. This imaging modality can be applied for the quality control of printed cDNA, oligonucleotide, and protein microarrays.