Abstract
An important application of robotically spotted DNA microarrays is the monitoring of RNA expression levels1,2. 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 reporters3,4. We have used a recently introduced luminescent reporter technology, called UPT (up-converting phosphor technology5,6). Down-converting phosphors have been applied before to detect nucleic acids on filters using time-resolved fluorometry7,8. The unique feature of the phosphor particles (size 0.4 μm) used here is that they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversion9,10. 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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Acknowledgements
The authors thank Mr. Victor de Jager for his contribution in preparing the expression arrays, Dr. Michael Bittner (NIH, Bethesda) for providing the cDNAs, and Dr. Johan den Dunnen (Department of Human Genetics, LUMC, Leiden) and Dr. Joe Gray (UCSF Cancer Center, San Francisco CA) for valuable discussions. Financially supported in part by the “Netherlands Organisation for Scientific Research” NWO grant nrs 901-01-208 and 925-01-008.
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van de Rijke, F., Zijlmans, H., Li, S. et al. Up-converting phosphor reporters for nucleic acid microarrays. Nat Biotechnol 19, 273–276 (2001). https://doi.org/10.1038/85734
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DOI: https://doi.org/10.1038/85734
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