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An important application of robotically spotted DNA microarrays is the monitoring of RNA expression levels. A clear limitation of this technology is the relatively large amount of RNA that is required per hybridization as a result of low hybridization efficiency and limiting detection sensitivity provided by conventional fluorescent reporters. We have used a recently introduced luminescent reporter technology, called UPT (up-converting phosphor technology). Down-converting phosphors have been applied before to detect nucleic acids on filters using time-resolved fluorometry. The unique feature of the phosphor particles (size 0.4 microm) used here is that they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversion. Because neither support material of microarrays nor biomolecules possess up-conversion properties, an enhanced image contrast is expected when these nonfading phosphor particles are applied to detect nucleic acid hybrids on microarrays. Comparison of the UPT reporter to cyanin 5 (Cy5) in a low-complexity model system showed a two order of maginitude linear relationship between phosphor luminescence and target concentration and resulted in an excellent correlation between the two reporter systems for variable target concentrations (R2 = 0.95). However, UPT proved to be superior in sensitivity, even though a wide-field microscope equipped with a xenon lamp was used. This higher sensitivity was demonstrated by complementary DNA (cDNA) microarray hybridizations using cDNAs for housekeeping genes as probes and complex cDNA as target. These results suggest that a UPT reporter technology in combination with a dedicated IR laser array-scanner holds significant promise for various microarray applications.

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The peroxidase-mediated deposition of hapten- and fluorochrome-labeled tyramides has recently been shown to increase the sensitivity of immunofluorescence and fluorescence in situ hybridization techniques. We have evaluated a number of red, green, and blue fluorescent tyramides for detection of antigens in tissue sections and cytospin preparations and for the detection of hapten- and horseradish peroxidase-labeled probes hybridized in situ to cells and chromosomes. With few exceptions, all fluorescent tyramide-based methods provided a considerable increase in sensitivity compared to conventional immunofluorescence and FISH methods.

To determine the presence of vascular endothelial growth factor A (VEGF-A) in the aqueous humor of eyes with uveal melanoma and to identify its source. Methods: The VEGF-A concentrations were determined in aqueous humor samples obtained after enucleation from 74 eyes with untreated uveal melanoma and from 8 eyes with treated uveal melanoma. Patient survival and clinical and histopathological tumor variables were compared. In situ hybridization, Western blot analysis, and enzyme-linked immunosorbent assay were used to determine expression of VEGF-A in tumor tissue and in overlying retina. Results: Aqueous VEGF-A concentrations ranged from 18 to 826 pg/mL in 74 untreated eyes, while concentrations in 30 control eyes were significantly lower (median, 50.1 pg/mL) (PϽ.001). Concentrations in 8 treated eyes were much higher (median, 364 pg/mL). In situ hybridization on tissue sections and Western blot analysis and enzyme-linked immunosorbent assay on tissue extracts revealed VEGF-A in uveal melanoma tissue and in retinal tissue. Conclusions: Uveal melanoma is associated with increased concentrations of VEGF-A in aqueous humor. Aqueous VEGF-A concentration correlates with largest basal tumor diameter and with the tumor height. In eyes with uveal melanoma, tumor and retinal tissues are sources of VEGF-A.