Signal amplification in the detection of single-copy DNA and RNA by enzyme-catalyzed deposition (CARD) of the novel fluorescent reporter substrate Cy3.29-tyramide

We demonstrate that the CAtalyzed Reporter Deposition method (CARD), utilizing the novel fluorescent reporter Cy3.29-tyramide, is successful in the Fluorescent In Situ Hybridization (FISH) detection of RNA and single-copy DNA. Histone 4 expression is detected in RNA extracts of S-phase, synchronized HeLa cells by dot-blot analysis. Gene expression of histone 4 in HeLa cells is demonstrated by FISH via CARD, utilizing oligonucleotide probes. Fluorescence intensity measurements on CARD-amplified histone 4 RNA detection showed (a) a 25-fold amplification of the signal brightness by biotinylated oligonucleotide probes and (b) a sixfold amplification of the signal brightness by horseradish peroxidase (HRP)-labeled histone 4 probes vs the directly stained control. The sensitivity of the CARD method is demonstrated by the FISH detection of single-copy DNA on human corneal fibroblast and HeLa 53 interphase nuclei. Chromosomal localization of the single copy DNA is demonstrated on HeLa 53 metaphase chromosome spreads.