Up-converting phosphor reporters for nucleic acid microarrays.
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.
Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Center; Leiden, The Netherlands.
F
F
van De Rijke
H
H
Zijlmans
S
S
Li
T
T
Vail
A K
AK
Raap
R S
RS
Niedbala
H J
HJ
Tanke
D019921:Biotinylation; D002232:Carbocyanines / Q000378:metabolism; D003001:Cloning, Molecular; D015342:DNA Probes / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007259:Infrared Rays; D008163:Luminescent Measurements; D015335:Molecular Probes; D009696:Nucleic Acids / Q000032:analysis / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D002232:Carbocyanines; D015342:DNA Probes; D018076:DNA, Complementary; D015335:Molecular Probes; D009696:Nucleic Acids; D012333:RNA, Messenger; C085321:cyanine dye 5