Accuracy of the microarray technology results is raised by using the multi-stage normalization of results. One of the principal requirements of such normalization is usage of internal standards. The routine Agilent microarray-based gene expression analysis protocol utilizes a Spike-In Kit during preparation of the samples representing a mixture of RNA fragments in different ratios. RNA probes which were synthesized in vitro conditions could be also used to establish how the magnitude of the fluorescent signal reflects the presence of RNA in the sample. A significant disadvantage of this type of standards is a difficulty of their production and the low RNA stability. In accordance with the Agilent protocol, the presence of the T7 promoter is necessary for the synthesis of labeled cRNA during sample preparation procedure. We hypothesized that we can successfully synthesize any RNA sequence having such type of promoter in its start position. Moreover, DNA sequence would serve as a matrix in this case. Using a set of different genes attached downstream of the T7-promoter in the plasmid DNA we have demonstrated in this study that such system can serve as a reliable template for the fluorescent labeled RNA sequence synthesis. In comparison with the routinely used internal RNA based controls, this template is stable, easy to manufacture and can be easily obtained in large quantities.
Download PDF:
Keywords: microarrays, plasmid DNA, T7-promoter, fluorescence level, labeled cRNA
Citation:
Kurbatov L.K., Zgoda V.G. (2016) A possibility to use the DNA-based probes as internal standards for Agilent Technologies microarray transcriptomic analysis. Biomeditsinskaya Khimiya, 62(6), 715-719.
Kurbatov L.K. et al. A possibility to use the DNA-based probes as internal standards for Agilent Technologies microarray transcriptomic analysis // Biomeditsinskaya Khimiya. - 2016. - V. 62. -N 6. - P. 715-719.
Kurbatov L.K. et al., "A possibility to use the DNA-based probes as internal standards for Agilent Technologies microarray transcriptomic analysis." Biomeditsinskaya Khimiya 62.6 (2016): 715-719.
Kurbatov, L. K., Zgoda, V. G. (2016). A possibility to use the DNA-based probes as internal standards for Agilent Technologies microarray transcriptomic analysis. Biomeditsinskaya Khimiya, 62(6), 715-719.
References
Dudley A.M., Aach J., Steffen M.A., Church G.M. (2002) Proc. Natl. Acad. Sci. USA, 99, 7554-7559. CrossRef Scholar google search
Carter M.G., Sharov A.A., VanBuren V., Dudekula D.B., Carmack C.E., Nelson C. et al. (2005) Genome Biology, 6, R61. Scholar google search
Kanno J., Aisaki K., Igarashi K., Nakatsu N., Ono A., Kodama Y., Nagao T. (2006) BMC Genomics, 7, p. 64. Scholar google search
Hill A.A., Brown E.L., Whitley M.Z., Tucker-Kellogg G., Hunter C.P., Slonim D.K. (2001) Genome Biology, 2, RESEARCH0055. Scholar google search
van de Peppel J., Kemmeren P., van Bakel H., Radonjic M., van Leenen D., Holstege F.C. (2003) EMBO Rep, 4, 387-393. CrossRef Scholar google search
Kakuhata R., Watanabe M., Yamamoto T., Akamine R., Yamazaki N., Kataoka M. et al. (2007) J. Biochem. Biophys. Methods, 70, 755-760. CrossRef Scholar google search
Hesse J., Jacak J., Kasper M., Regl G., Eichberger T., Winklmayr M., Aberger F., Sonnleitner M., Schlapak R., Howorka S., Muresan L., Frischauf A.M., Schutz G.J. (2006) Genome Res., 16, 1041-1045. CrossRef Scholar google search
Kakuhata R., Watanabe M., YamamotoT., Obana E., Yamazaki N., Kataoka M., Ooie T., Baba Y., Hori T., Shinohara Y. (2008) J. Biochem. Biophys. Methods, 70, 926-931. CrossRef Scholar google search
