Lee Myoyong, Trent Jeffrey M, Bittner Michael L
Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Anal Biochem. 2007 Sep 1;368(1):61-9. doi: 10.1016/j.ab.2007.06.005. Epub 2007 Jun 8.
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.
目前，DNA微阵列所提供的测量质量足够高，能够得出关于基因调控以及细胞过程协调作用的各种可靠推断。尽管如此，对测量更高精度的需求仍促使微阵列研究群体通过改进阵列制备、样本标记和杂交技术来追求更高的测量质量。如先前一项研究所描述的，我们通过在醛基官能团衍生化的载玻片上印刷65聚体来制备寡核苷酸微阵列。我们可以通过在探针标记过程中消除酶促偏差并在更严格的条件下杂交来提高数据的可靠性。这种优化方法被用于分析九种不同小鼠组织和器官的基因表达模式，数据的多维尺度分析（MDS）表明，同类样本之间具有很强的相似性，不同组织样本之间有清晰、高度可重复的区分。另外三种微阵列是在商业基质上制备的，并按照制造商的说明进行杂交。然后将这些数据与自制微阵列数据以及逆转录-聚合酶链反应（RT-PCR）数据进行比较。就信号强度、背景和杂交特性而言，定制醛基载玻片上印刷的微阵列优于商业可用基质载玻片上印刷的微阵列。定制基质微阵列的数据在转录本丰度变化高达100倍的定量变化方面通常与RT-PCR数据显示出良好的一致性。然而，随着公共数据领域收集到更多的基因组序列信息，将会进行更准确的比较。
