Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples

Abstract

DNA or oligonucleotide arrays are widely used for large-scale expression measurements, using various implementations: macroarrays in which DNA is spotted onto nylon membranes of relatively large dimensions (with radioactive detection) on the one hand; microarrays on glass slides and oligonucleotide chips, both used with fluorescent probes, on the other hand. Nylon micro-arrays with colourimetric detection have also been described recently. The small physical dimensions of miniaturized systems allow small hybridization volumes (2-100 microl) and provide high probe concentrations, in contrast to macroarrays. We show, however, that actual sensitivity (defined as the amount of sample necessary for detection of a given mRNA species) is in fact similar for all these systems and that this is mostly due to the very different amounts of target material present on the respective arrays. We then demonstrate that the combination of nylon microarrays with(33)P-labelled radioactive probes provides 100-fold better sensitivity, making it possible to perform expression profiling experiments using submicrogram amounts of unamplified total RNA from small biological samples. This has important implications in basic and clinical research and makes this alternative approach particularly suitable for groups operating in an academic context.