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

DNA or oligonucleotide arrays are widely used for large-scale expression me asurements, using various implementations: macroarrays in which DNA is spot ted onto nylon membranes of relatively large dimensions (with radioactive d etection) on the one hand; microarrays on glass slides and oligonucleotide chips, both used with fluorescent probes, on the other hand. Nylon microarr ays with colourimetric detection have also been described recently. The sma ll physical dimensions of miniaturized systems allow small hybridization vo lumes (2-100 mu l) and provide high probe concentrations, in contrast to ma croarrays. We show, however, that actual sensitivity (defined as the amount of sample necessary for detection of a given mRNA species) is in fact simi lar for ail these systems and that this is mostly due to the very different amounts of target material present on the respective arrays. We then demon strate that the combination of nylon microarrays with P-33-labelled radioac tive probes provides 100-fold better sensitivity, making it possible to per form expression profiling experiments using submicrogram amounts of unampli fied total RNA from small biological samples. This has important implicatio ns in basic and clinical research and makes this alternative approach parti cularly suitable for groups operating in an academic context.