Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays.
Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays.
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DOI：
10.1186/1471-2164-5-12
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发表时间：
2004-02-09
期刊：
影响因子：
4.4
通讯作者：
Zahrt TC
中科院分区：
文献类型：
--
作者：
Hessner MJ;Singh VK;Wang X;Khan S;Tschannen MR;Zahrt TC
斑点70-mer寡核苷酸阵列提供了潜在的更高的特异性和替代昂贵的cDNA文库维护和扩增。由于微阵列制造是数据差异的重要来源，我们之前直接用第三个荧光团标记cDNA探针进行预杂交质量控制。荧光修饰寡核苷酸组的成本过高，因此，一种共同斑点的金黄色葡萄球菌特异性荧光标记的“跟踪”寡核苷酸被描述为监测结核分枝杆菌寡核苷酸微阵列的制造变量。与供应商推荐的缓冲液相比，在15% DMSO/1.5 M甜菜碱中打印的DNA保留率显著提高（p < 0.01）。在结核分枝杆菌H37Rv和结核分枝杆菌mprA杂交中，引入跟踪寡核苷酸不影响杂交效率或引入比值测量偏差。具有或不具有跟踪寡核苷酸的阵列差异表达基因的平均对数Cy3/Cy5比值呈线性关系（R2 = 0.90, p < 0.05），具有（0.72±0.07）、不具有（0.74±0.10）或不具有（0.70±0.04）跟踪寡核苷酸的重复之间的比值数据的Pearson相关系数无显著差异。方差分析证实跟踪寡核苷酸没有引入偏倚。在0.5 μM跟踪寡核苷酸存在的情况下，滴定靶特异性寡核苷酸（40 μM至0.78 μM），发现荧光素荧光与靶特异性寡核苷酸的量浓度呈负相关，使跟踪寡核苷酸信号有助于质量控制测量和区分假阴性（合成失败和机械失误）和真阴性（无基因表达）。这种新颖的方法使斑点寡核苷酸阵列的预杂交阵列可视化，并为更复杂的玻片鉴定和数据过滤应用奠定了基础。
Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray. Significantly (p < 0.01) improved DNA retention was achieved printing in 15% DMSO/1.5 M betaine compared to the vendor recommended buffers. Introduction of tracking oligonucleotide did not effect hybridization efficiency or introduce ratio measurement bias in hybridizations between M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p < 0.05) and there were no significant differences in Pearson's correlation coefficients of ratio data between replicates possessing (0.72 ± 0.07), replicates lacking (0.74 ± 0.10), or replicates with and without (0.70 ± 0.04) the tracking oligonucleotide. ANOVA analysis confirmed the tracking oligonucleotide introduced no bias. Titrating target-specific oligonucleotide (40 μM to 0.78 μM) in the presence of 0.5 μM tracking oligonucleotide, revealed a fluorescein fluorescence inversely related to target-specific oligonucleotide molarity, making tracking oligonucleotide signal useful for quality control measurements and differentiating false negatives (synthesis failures and mechanical misses) from true negatives (no gene expression). This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.
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