Genomic DNA as a General Cohybridization Standard for Ratiometric Microarrays

Abstract

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.

Additional details