Sensitivity Issues in DNA Array-Based Expression Measurements and Performance of Nylon Microarrays for Small Samples

TLDR

DNA and oligonucleotide arrays, including macroarrays on nylon membranes and microarrays on glass slides, are widely used for large‑scale expression profiling, and miniaturized nylon microarrays with colourimetric detection allow small hybridization volumes and high probe concentrations. We found that overall sensitivity is similar across array types, but combining nylon microarrays with 33P‑labelled probes yields a 100‑fold increase, enabling expression profiling from sub‑microgram unamplified RNA and benefiting small‑sample studies in academic settings.

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 microarrays with colourimetric detection have also been described recently. The small physical dimensions of miniaturized systems allow small hybridization volumes (2–100 µl) 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 33P-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.