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.

Gene expression profiling identifies molecular subgroups among nodal peripheral T-cell lymphomas

“…Data were further corrected for the amount of spotted cDNA (Bertucci et al, 1999). From the initial set of 7.824 genes related to carcinogenesis, 4.716 were significantly expressed in a majority of samples and were kept for further analysis.…”

“…Several quality control steps were taken. Before each RNA hybridization, the quality of spotting, including the determination of target DNA amount accessible for each spot, was controlled by hybridization of each microarray with a 33 P-labeled oligonucleotide sequence common to all PCR products (Bertucci et al, 1999). After stripping, microarrays were hybridized with 33 P-labeled probes made from total RNA.…”

High Cytotoxic and Specific Migratory Potencies of Senescent CD8+CD57+ Cells in HIV-Infected and Uninfected Individuals

“…A-MEXP-50 and E-MEXP-393; ͗www.ebi.ac.uk/miamexpress͘). The feasibility, reproducibility, and sensitivity of spotting procedures have been previously described (25)(26)(27). Nylon microarrays were prepared as described using 3277 IMAGE cDNA clones (3158 nonredundant) coding for genes involved in the immunological process and ubiquitous biological function (26).…”

Distinct and Complementary Information Provided by Use of Tissue and DNA Microarrays in the Study of Breast Tumor Markers

“…Nylon filter preparation with spotted polymerase chain reaction (PCR) products derived from ϳ1000 selected candidate cancer genes, 33 P radioactive hybridization, and data acquisition, normalization, and analysis have been described elsewhere 13,14 and can also be consulted on our web site (http:/tagc.univ-mrs.fr/pub/Cancer/). …”