Evaluation of the performance of two carbodiimide-based cyanine dyes for detecting changes in mRNA expression with DNA microarrays
Evaluation of the performance of two carbodiimide-based cyanine dyes for detecting changes in mRNA expression with DNA microarrays
复制标题
DOI：
10.2144/05385mt02
复制
发表时间：
2005-05-01
期刊：
影响因子：
2.7
通讯作者：
Murakami, M
中科院分区：
文献类型：
--
作者：
Kimura, N;Tamura, TA;Murakami, M
微阵列已被广泛用于研究全基因组表达模式。虽然这项技术已经取得了巨大的成功，一些实际问题将受益于设计的改进。在这里，我们描述了一种新的，有效的标记方法，使用双酰亚胺连接的花青染料直接化学标记来自小鼠总RNA的cDNA。使用该方案，在70 ℃下仅需10分钟即可完成cDNA标记反应。直接标记的cDNA然后可以与70-mer小鼠寡核苷酸阵列杂交用于表达谱研究。微阵列分析表明，这些cDNA被均匀地标记，并产生更高的信号强度比传统的酶直接标记方法和可比的信号强度，通过传统的间接标记方法获得的。此外，验证我们的微阵列数据使用逆转录PCR（RT-PCR）方法表明两种方法之间的良好协议。因此，我们的结论是，我们的简化花青-碳二亚胺标记方法，它不依赖于改性核苷酸的掺入，将提供一个可靠的，更快的和潜在的更便宜的替代既定的标记技术的基因表达分析。
Microarrays have been extensively used to investigate genome-wide expression patterns. Although this technology has been tremendously successful, several practical issues would benefit from improvements in design. Here we describe a novel, efficient labeling methodology that uses carbodiimide-linked cyanine dyes to directly chemically label cDNA derived from mouse total RNA. Using this protocol, it takes only 10 min at 70 degrees C to complete the cDNA labeling reaction. The directly labeled cDNAs can then be hybridized to 70-mer mouse oligonucleotide arrays for expression profiling studies. Microarray analyses indicate that these cDNAs are uniformly labeled and produce higher signal intensities than conventional enzymatic direct labeling methods and comparable signal intensities to those obtained by conventional indirect labeling methods. Furthermore, verification of our microarray data using a reverse transcription PCR (RT-PCR) method indicates good agreement between the two methods. Thus, we conclude that our simplified cyanine-carbodiimide labeling method, which does not rely on the incorporation of modified nucleotides, will provide a reliable, quicker and potentially cheaper alternative to established labeling techniques for gene expression analyses.