Adequate Design of Customized cDNA Macroarray for Convenient Multiple Gene Expression Analysis(METHOD)

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概要

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• To establish a convenient, cost-effective, and reasonably reliable method for monitoring multiple gene expression using customized membrane-based macroarray, we constructed a cDNA macroarray with multiple probes for 13 human vascular endothelial genes and assessed the accuracy of the macroarray measurements. For each gene, two cDNA probes (450-550bp) were designed from different regions (coding region and 3'-untranslated region [3'-UTR], respectively) on the basis of simple criteria concerning length and sequence specificity and spotted on the macroarray. In addition, unmodified oligonucleotide probes (80 mer) targeted to a unique sequence from the coding region of each gene were spotted on the same macroarray. Using this macroarray, shear stress-induced mRNA expression changes were analyzed in human coronary artery endothelial cells. Comparison of the expression ratios obtained with those measured using quantitative realtime polymerase chain reaction (PCR) as a reference method revealed that cDNA probes designed from a sequence within the coding region provided a highly accurate expression profile, whereas results obtained from oligonucleotide probes showed no correlation with real-time PCR data, which might be caused by inadequate immobilization of oligonucletotide probes on the nylon membrane. In addition, we observed that cDNA probes targeting different regions of a gene yielded different signal intensities. Most cDNA probes designed from a sequence within the coding region showed detectable signals, whereas few cDNA probes designed from 3'-UTR did.

• 社団法人日本生物工学会の論文
• 2007-01-25

著者

• Ando Joji

  Department of Cardiovascular Biomechanics, Faculty of Medicine, University of Tokyo

• Ando Joji

  獨協医科大学 医学部生体医工学

• YAMAMOTO Kimiko

  Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo

• Yamamoto Kimiko

  Department Of Biomedical Engineering Graduate School Of Medicine University Of Tokyo

• Ando Joji

  Department Of Biomedical Engineering Faculty Of Medicine University Of Tokyo

• Nakagawa Miki

  Lab Co. Ltd.

• Sugiyama Shunpei

  Lab Co., Ltd.

• Nishimura Norihiro

  Medical Industrial Linkage Office, Graduate School of Medicine, Mie University

• Maruta Yukio

  Lab Co., Ltd.

• Maruta Yukio

  Lab Co. Ltd.

• Sugiyama Shunpei

  Lab Co. Ltd.

• Nishimura Norihiro

  Medical Industrial Linkage Office Graduate School Of Medicine Mie University

• Ando Joji

  Lab. Of Biomedical Engineering School Of Medicine Dokkyo Medical Univ. Jpn

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