Use of RNA and genomic DNA references for inferred comparisons in DNA microarray analyses

In most microarray assays, labeled cDNA molecules derived from reference and query RNA samples are co-hybridized to probes arrayed on a glass surface. Gene expression profiles are then calculated for each gene based on the relative hybridization intensities measured between the two samples. The most commonly used reference samples are typically isolates from a single representative RNA source (RNA-0) or pooled mixtures of RNA derived from a plurality of sources (RNA-p). Genomic DNA offers an alternative reference nucleic acid with a number of potential advantages, including stability, reproducibility, and a potentially uniform representation of all genes, as each unique gene should have equal representation in a haploid genome. Using hydrogen peroxide-treated Arabidopsis thaliana plants as a model, we evaluated genomic DNA and RNA-p as reference samples and compared expression levels inferred through the reference relative to unexposed plants with expression levels measured directly using an RNA-0 reference. Our analysis demonstrates that while genomic DNA can serve as a reasonable reference source for microarray assays, a much greater correlation with direct measurements can be achieved using an RNA-based reference sample.