Genomic DNA as a cohybridization standard for mammalian microarray measurements.
A persistent design problem for ratiometric microarray studies is selecting the 'denominator' RNA cohybridization standard. The ideal standard should be readily available, inexpensive, invariant over time and from laboratory to laboratory, and should represent all genes with a uniform signal. RNA references (both commercial 'universal' and experiment--specific types), fall short of these goals. We show here that mouse genomic DNA is a reliable microarray cohybridization standard which can meet these criteria. Genomic DNA was superior in universality of coverage (>98% of genes from a 16,000 feature mouse 70mer microarray) to the Stratagene Universal Mouse Reference RNA standard. Ratios for genes in very low abundance in the Stratagene standard were more unstable with the Stratagene standard than with genomic DNA. Genes with mid-range, and therefore presumably optimal RNA denominator values, showed comparable reproducibility with both standards. Inferred ratios made between two different experimental RNAs using a genomic DNA standard were found to correlate well with companion, directly measured ratios (Spearman correlation coefficient = 0.98). The advantage in array feature coverage of genomic DNA will likely increase as newer generation microarrays include genes which are expressed exclusively in minor tissue or developmental domains that are not represented in mixed tissue RNA standards.
Williams
Brian A
BA
Division of Biology, MC 156-29, California Institute of Technology, Pasadena, CA 91125, USA.
Gwirtz
Richele M
RM
Wold
Barbara J
BJ
D000818:Animals; D017360:Arabidopsis / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016678:Genome; D008322:Mammals / Q000235:genetics; D051379:Mice; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results
D004247:DNA