Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes.
Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes.
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DOI：
10.1186/1471-2164-10-392
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发表时间：
2009-08-22
期刊：
影响因子：
4.4
通讯作者：
Kiyosawa H
中科院分区：
文献类型：
--
作者：
Numata K;Osada Y;Okada Y;Saito R;Hiraiwa N;Nakaoka H;Yamamoto N;Watanabe K;Okubo K;Kohama C;Kanai A;Abe K;Kiyosawa H
最近的转录组学分析在哺乳动物中发现了广泛存在的内源性反义转录物，称为天然反义转录物（NAT）。NAT从基因位点的相反链转录，被认为控制有义基因的表达，但这种调节的机制尚不清楚。尽管在哺乳动物中已经鉴定出数千个潜在的正义-反义对，但功能特征的NAT的实例仍然有限。为了确定NAT候选人适合进一步的功能分析，我们进行了基于DNA微阵列的NAT筛选小鼠成年正常组织和乳腺肿瘤的目标不仅是有义方向，但也注释基因的互补链。首先，我们设计了微阵列探针，以靶向基因的互补链，其反义对应物仅在人类公共cDNA来源中被确定，但在小鼠中未被确定。我们观察到66.1%的635个靶基因的显著表达信号，其中58个基因表现出组织特异性表达。对所选实例（Escha 1b和Aard）的表达分析证实了它们在体内的动态转录。虽然种间保护NAT表达的存在下，在这两个物种的cDNA来源，我们的研究结果表明，有更多的例子，人-小鼠保守的NAT，不能确定的cDNA来源。我们还设计了探针以靶向包括癌基因在内的特征基因的互补链，并比较了乳腺癌组织和非病理组织之间这些基因的表达。我们发现，404个注释良好的基因中有95个基因的反义表达在肿瘤组织中与正常组织相比发生了显着变化，其中19个基因的正义基因表达也发生了变化。这些结果突出了NAT表达在细胞事件调节和病理条件中的重要性。我们的微阵列平台靶向注释基因的互补链，成功地鉴定了新的NAT，这些NAT不能通过实验室可用的cDNA数据来鉴定，因此不能通过通常的“有义靶向”微阵列方法来检测。通过该平台监测的差异表达的NAT可以为基因功能的研究提供候选者。我们的微阵列平台的一个优点是它可以应用于任何感兴趣的基因和靶样品。
Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs). NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes. First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard) confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation of cellular events and in pathological conditions. Our microarray platform targeting the complementary strand of annotated genes successfully identified novel NATs that could not be identified by publically available cDNA data, and as such could not be detected by the usual "sense-targeting" microarray approach. Differentially expressed NATs monitored by this platform may provide candidates for investigations of gene function. An advantage of our microarray platform is that it can be applied to any genes and target samples of interest.
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