Three color cDNA microarrays: quantitative assessment through the use of fluorescein-labeled probes
Three color cDNA microarrays: quantitative assessment through the use of fluorescein-labeled probes
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DOI：
10.1093/nar/gng014
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发表时间：
2003-02-15
影响因子：
14.9
通讯作者：
Ghosh, S
中科院分区：
文献类型：
--
作者：
Hessner, MJ;Wang, XJ;Ghosh, S
使用微阵列的基因表达研究具有巨大的潜力，可以为人类疾病发病机制提供新的见解，但数据质量仍然是一个主要障碍。特别是，在良好的研究设计和靶标制备的情况下，不存在一种方法可以在杂交之前确定阵列是否会产生高质量的数据。我们通过开发三色 cDNA 微阵列平台解决了这个问题，其中打印的探针采用荧光素标记，但在使用具有窄带宽的共焦激光扫描仪时与 Cy3 和 Cy5 染料标记的靶标在光谱上兼容。该方法能够对阵列/斑点形态、DNA 沉积和保留以及背景水平进行预杂交评估。通过使用这些测量值和载玻片内荧光强度的变异系数，我们表明同一批次中的载玻片并不等效，并且可测量的预杂交参数可以预测由重复一致性确定的杂交性能。当将来自两个细胞系的靶标与高质量和低质量重复对（n = 50 对）杂交时，观察到杂交前信号与背景噪声和杂交后重现性之间存在直接且显着的关系（R-2 = 0.80，P < 0.001）。因此，我们得出结论，基于预杂交质量评分的载玻片选择将极大地有利于生成可靠的基因表达数据的能力。
Gene expression studies using microarrays have great potential to generate new insights into human disease pathogenesis, but data quality remains a major obstacle. In particular, there does not exist a method to determine prior to hybridization whether an array will yield high quality data, given good study design and target preparation. We have solved this problem through development of a three-color cDNA microarray platform where printed probes are fluorescein labeled, but are spectrally compatible with Cy3 and Cy5 dye-labeled targets when using confocal laser scanners possessing narrow bandwidths. This approach enables prehybridization evaluation of array/spot morphology, DNA deposition and retention and background levels. By using these measurements and the intra-slide coefficient of variation for fluorescence intensity we show that slides in the same batch are not equivalent and measurable prehybridization parameters can be predictive of hybridization performance as determined by replicate consistency. When hybridizing target derived from two cell lines to high and low quality replicate pairs (n = 50 pairs), a direct and significant relationship between prehybridization signal-to-background noise and post-hybridization reproducibility (R-2 = 0.80, P < 0.001) was observed. We therefore conclude that slide selection based upon prehybridization quality scores will greatly benefit the ability to generate reliable gene expression data.