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Chapter 45 : Principles and Practice of DNA Microarray Technology

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Abstract:

This chapter describes the principles and detailed methodology for carrying out DNA microarray experiments using the yeast , and briefly reviews its attendant applications. Although microarray technology is widely used to study the transcriptome and its differential expression, this technology has also been applied in novel ways to study various aspects of cell biology. The two RNA samples, one derived from experimental RNA and the other from the control RNA sample, are converted to cDNA and labeled individually using fluorescent dyes such as cyanin 5 (Cy5) and cyanin 3 (Cy3). Next, the two labeled samples (targets) are combined in equal proportions and hybridized to a single microarray slide, therefore referred to as dual-color hybridization, involving competitive hybridization, i.e., simultaneous hybridization of the reference and experimental targets to a single microarray. The fluorescent signal emanating from each feature on the microarray slide is quantified by scanning the slide in a microarray scanner. The ratio of the fluorescence intensities emanating from the experimental versus control samples is then subjected to extensive statistical analyses to obtain the relative expression level of each gene on the microarray. A careful interpretation of the microarray data is important so that meaningful hypotheses can be generated for designing follow-up experiments. Further refinement of the statistical tools for microarray data analysis, including standardization of data normalization methods, would yield more robust and reliable data and enable comparison of microarray data generated in different laboratories and possibly find use in clinical practice.

Citation: Natarajan K, Marton M, Hinnebusch A. 2007. Principles and Practice of DNA Microarray Technology, p 978-994. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch45

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DNA Synthesis
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RNA Polymerase II
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FIGURE 1

Overview of DNA microarray scheme.

Citation: Natarajan K, Marton M, Hinnebusch A. 2007. Principles and Practice of DNA Microarray Technology, p 978-994. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch45
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Image of FIGURE 2
FIGURE 2

Suggested workflow for comprehensive assessment of genome function. Integration of results from microarray studies into the results of proteome analysis, metabolome analysis, and protein-protein interaction data would provide a broad platform for assignment of genes to respective pathways and inference of the biological function of novel genes.

Citation: Natarajan K, Marton M, Hinnebusch A. 2007. Principles and Practice of DNA Microarray Technology, p 978-994. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch45
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