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Genomic DNA as a General Cohybridization Standard for Ratiometric Microarrays

Williams, Brian A. and Gwirtz, Richele M. and Wold, Barbara J. (2006) Genomic DNA as a General Cohybridization Standard for Ratiometric Microarrays. In: DNA Microarrays, Part A: Array Platforms and Wet-Bench Protocols. Methods in Enzymology. No.410. Academic Press , Burlington, MA, pp. 237-279. ISBN 9780121828158.

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Feature variability on ratiometric microarrays is accommodated by simultaneous cohybridization of a labeled reference standard with a labeled experimental sample. An optimal reference standard would provide full and equal representation for all array features from a given genome so that it would function on any array, would represent all features with similar signal intensity, and would be highly reproducible—both technically and biologically—from preparation to preparation and laboratory to laboratory. A low cost and a good shelf life are also highly desirable. Finally, providing for straightforward recovery of RNA prevalence information and for integration of data across multiple, initially unrelated studies would be significant advances over current methods. For virtually all ratiometric array studies published to date the reference standard has been some kind of RNA sample assembled from a number of different cell lines, tissues, or experimental time points. These RNA references fall short of the desired universality, uniformity, and reproducibility criteria, which then affect data quality and integration across studies. Also, the various mixed RNA standards cannot be used to derive RNA prevalence information from an experimental sample. In contrast, genomic DNA is a natural choice to meet all the criteria, although it has not yet been widely exploited for eukaryotic array experiments. Principal stumbling blocks have been achieving high enough absolute signals for large mammalian and plant genomes and finding a way to stabilize labeled DNA so that it can be stored and used with ease. This chapter describes two genomic DNA‐labeling methods that make it possible to use genomic DNA as a universal microarray cohybridization standard. The indirect labeling method permits production of a large quantity of a stable genomic DNA standard that can then be quality tested and stored frozen. This optimizes experimental consistency and significantly improves ease of use. This chapter also shows that the genomic DNA reference standard can deliver RNA prevalence measurements from ratiometric array platforms.

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Wold, Barbara J.0000-0003-3235-8130
Additional Information:© 2006, Elsevier Inc. We are indebted to our Bioinformatics staff (Diane Trout, Brandon King, and Joe Roden) for their patient assistance with this project. This work was funded by grants to BJW from the Department of Energy (DOE), the National Aeronautic and Space Administration (NASA), and the National Institutes of Health (NIH). BAW was supported by an NIH NRSA fellowship.
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Department of Energy (DOE)UNSPECIFIED
NIH Predoctoral FellowshipUNSPECIFIED
Series Name:Methods in Enzymology
Issue or Number:410
Record Number:CaltechAUTHORS:20190507-155913409
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Official Citation:Brian A. Williams, Richele M. Gwirtz, Barbara J. Wold, [12] Genomic DNA as a General Cohybridization Standard for Ratiometric Microarrays, Methods in Enzymology, Academic Press, Volume 410, 2006, Pages 237-279, ISSN 0076-6879, ISBN 9780121828158, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:95327
Deposited By: Tony Diaz
Deposited On:07 May 2019 23:09
Last Modified:16 Nov 2021 17:11

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