Importance of probe location for quantitative comparison of signal intensities among genes in microarray analysis
Importance of probe location for quantitative comparison of signal intensities among genes in microarray analysis
复制标题
DOI：
10.1016/j.jprot.2007.12.005
复制
发表时间：
2008-04-24
影响因子：
--
通讯作者：
Shinohara, Yasuo
中科院分区：
文献类型：
--
作者：
Kakuhata, Rei;Watanabe, Masahiro;Shinohara, Yasuo
在我们以前的研究中，我们证明了通过Agilent寡核苷酸阵列系统（代码G4130 A）确定的基因的表达水平可以通过掺入合成的全长mRNA作为标准来定量评估[Kakuhata R，Watanabe M，Yamamoto T，Akamine R，Yamazaki N，Kataoka M，等，体外合成的特定组织表达的mRNA作为定量评价肿瘤细胞的标准的可能性，微阵列分析的结果，J Biochem Biophys Methods 2007;70：755-60]。然而，在其后继版本（Agilent oligo array system，代码G4131 F）中，其被建立以使得能够在更宽的动态范围内进行基因表达分析，多个探针通常用于评估单个基因的表达水平;并且它们显示出明显不同的信号强度。这一结果表明，在这个新版本中观察到的信号强度似乎并不简单地反映单个基因的转录水平。为了了解影响探针信号强度的因素，我们表征了这种新阵列系统中使用的探针的性质和标记过程中形成的cRNA。对与阵列杂交的反应混合物中的cRNA的分析显示，cRNA在所使用的条件下没有完全转录。因此，探测器位于。发现信使5'端的信号比3'端的信号低，并且观察到的信号强度取决于探针在mRNA中的位置。大量探针的mRNA上的信号强度和位置之间的相关性的分析也支持探针位置是微阵列分析中探针信号强度的主要决定因素之一的观点。(c)2008年由Elsevier B. V.出版。
In our previous studies, we demonstrated that expression levels of genes determined by Agilent oligoarray system, code G4130A, could be quantitatively evaluated by spike-in of synthetic full-length mRNAs as standards [Kakuhata R, Watanabe M, Yamamoto T, Akamine R, Yamazaki N, Kataoka M, et al. Possible utilization of in vitro synthesized mRNAs specifically expressed in certain tissues as standards for quantitative evaluation of the results of microarray analysis, J Biochem Biophys Methods 2007;70:755-60]. However, in its successor version (Agilent oligo array system, code G4131F), which was established to enable gene expression analysis over a much wider dynamic range, multiple probes are often utilized for evaluation of expression levels of individual genes; and they showed markedly distinct signal intensities. This result indicates that the observed signal intensities in this new version seemed not to simply reflect the transcript levels of individual genes. To understand the factors influencing the signal intensities of probes, we characterized the properties of the probes used in this new array system and the cRNAs formed during the labeling process. Analysis of cRNAs in the reaction mixture, which were hybridized with the arrays, revealed that the cRNAs were not fully transcribed under the conditions used. For this reason, probes located at. the 5' side of the message were found to give lower signals than those at the 3' end; and the observed signal intensities were dependent upon the location of probes in the mRNA. Analysis of the correlation between signal intensities and locations on mRNAs for larger numbers of probes also supported the idea that probe location is one of the major determinants of signal intensities of probes in microarray analysis. (c) 2008 Published by Elsevier B.V.