Optimization of oligonucleotide microarray fabricated by spotting 65-mer
Optimization of oligonucleotide microarray fabricated by spotting 65-mer
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DOI：
10.1016/j.ab.2007.06.005
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发表时间：
2007-09-01
影响因子：
2.9
通讯作者：
Bittner, Michael L.
中科院分区：
文献类型：
--
作者：
Lee, Myoyong;Trent, Jeffrey M.;Bittner, Michael L.
DNA微阵列目前提供了足够高质量的测量，以允许对基因调控和细胞过程的协调进行各种各样的合理推断。尽管如此，对测量更高精度的期望继续推动微阵列研究界通过改进阵列制造和样品标记和杂交来寻求更高的测量质量。我们制备寡核苷酸微阵列印刷65聚体上醛官能团衍生的载玻片，如在以前的研究中所描述的。我们可以通过消除探针标记过程中的酶偏差和在更严格的条件下进行杂交来提高数据的可靠性。这种优化的方法被用来分析9种不同小鼠组织和器官的基因表达模式，多维标度（MDS）数据分析显示，相似样品之间具有很强的相似性，不同组织样品之间具有清晰、高度可重复的分离。在商业基底上制造三个其他微阵列，并按照制造商的说明进行杂交。然后将这些数据与内部微阵列数据和逆转录聚合酶链反应（RT-PCR）数据进行比较。在信号强度、背景和杂交特性方面，印刷在定制醛载玻片上的微阵列上级印刷在市售基底载玻片上的微阵列。来自定制底物微阵列的数据通常显示出与RT-PCR数据在转录物丰度的高达100倍变化的定量变化方面的良好一致性。然而，随着更多的基因组序列信息在公共数据域中收集，将进行更准确的比较。爱思唯尔公司出版
DNA microarrays currently provide measurements of sufficiently high quality to allow a wide variety of sound inferences about gene regulation and the coordination of cellular processes to be drawn. Nonetheless, a desire for greater precision in the measurements continues to drive the microarray research community to seek higher measurement quality through improvements in array fabrication and sample labeling and hybridization. We prepared oligonucleotide microarrays by printing 65-mer on aldehyde functional group-derivatized slides as described in a previous study. We could improve the reliability of data by removing enzymatic bias during probe labeling and hybridizing under a more stringent condition. This optimized method was used to profile gene expression patterns for nine different mouse tissues and organs, and multidimensional scaling (MDS) analysis of data showed both strong similarity between like samples and a clear, highly reproducible separation between different tissue samples. Three other microarrays were fabricated on commercial substrates and hybridized following the manufacturer's instructions. The data were then compared with in-house microarray data and reverse transcription-polymerase chain reaction (RT-PCR) data. The microarray printed on the custom aldehyde slide was superior to microarrays printed on commercially available substrate slides in terms of signal intensities, background, and hybridization characteristics. The data from the custom substrate microarray generally showed good agreement in quantitative changes up to 100-fold changes of transcript abundance with RT-PCR data. However, more accurate comparisons will be made as more genomic sequence information is gathered in the public data domain. Published by Elsevier Inc.