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在包被的聚合物载玻片上通过寡核苷酸微阵列进行表达谱分析。

Expression profiling by oligonucleotide microarrays spotted on coated polymer slides.

作者信息

Nielsen Peter S, Ohlsson Helle, Alsbo Carsten, Andersen Morten S, Kauppinen Sakari

机构信息

Department of Functional Genomics, Exiqon, Bygstubben 9, DK-2950 Vedbaek, Denmark.

出版信息

J Biotechnol. 2005 Mar 16;116(2):125-34. doi: 10.1016/j.jbiotec.2004.10.009. Epub 2004 Dec 8.

Abstract

We have developed a ready-to-spot polymer microarray slide, which is coated with a uniform layer of reactive electrophilic groups using anthraquinone-mediated photo-coupling chemistry. The slide coating reduces the hydrophobicity of the native polymer significantly, thereby enabling robust and efficient one-step coupling of spotted 5' amino-linked oligonucleotides onto the polymer slide. The utility of the coated polymer slide in gene expression profiling was assessed by fabrication of spotted oligonucleotide microarrays using a collection of 5' amino-linked 70-mer oligonucleotide probes representing 96 yeast genes from Operon. Two-colour hybridizations with labelled cDNA target pools derived from standard grown and heat-shocked wild type yeast cells could reproducibly measure heat shock induced expression of seven different heat shock protein (HSP) genes. Moreover, the observed fold changes were comparable to those reported previously using spotted cDNA arrays and high-density 25-mer oligonucleotide arrays from Affymetrix. The low hybridization signals obtained from the DeltaSSA4 mutant cDNA target, together with the high signal detected in two-colour hybridizations with heat-shocked wild type yeast relative to the DeltaSSA4 mutant strain implies that unspecific binding of cDNA target to the SSA4-specific 70-mer oligonucleotide probes is negligible. Combined, our results indicate that the coated polymer microarray slide represents a robust and cost-effective array platform for pre-spotted oligonucleotide arrays.

摘要

我们开发了一种即用型聚合物微阵列载玻片,它使用蒽醌介导的光偶联化学方法涂覆有一层均匀的反应性亲电基团。载玻片涂层显著降低了天然聚合物的疏水性,从而能够将点样的5'氨基连接的寡核苷酸稳健且高效地一步偶联到聚合物载玻片上。通过使用一组代表来自Operon的96个酵母基因的5'氨基连接的70聚体寡核苷酸探针制造点样寡核苷酸微阵列,评估了涂覆聚合物载玻片在基因表达谱分析中的实用性。与来自标准培养和热激野生型酵母细胞的标记cDNA靶标池进行双色杂交,可以重现性地测量七种不同热休克蛋白(HSP)基因的热激诱导表达。此外,观察到的倍数变化与先前使用点样cDNA阵列和Affymetrix的高密度25聚体寡核苷酸阵列报道的结果相当。从DeltaSSA4突变体cDNA靶标获得的低杂交信号,以及与DeltaSSA4突变体菌株相比,在与热激野生型酵母的双色杂交中检测到的高信号,意味着cDNA靶标与SSA4特异性70聚体寡核苷酸探针的非特异性结合可以忽略不计。综合来看,我们的结果表明,涂覆聚合物微阵列载玻片代表了一种用于预点样寡核苷酸阵列的稳健且经济高效的阵列平台。

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