title: 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.
abstract: 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 5-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 S5 interphase nuclei. Chromosomal localization of the single copy DNA is demonstrated on HeLa S3 metaphase chromosome spreads.
authors.affiliation: Center for Light Microscope, Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: B F
authors.initials: BF
authors.lastname: Schmidt
authors.forename: J
authors.initials: J
authors.lastname: Chao
authors.forename: Z
authors.initials: Z
authors.lastname: Zhu
authors.forename: R L
authors.initials: RL
authors.lastname: DeBiasio
authors.forename: G
authors.initials: G
authors.lastname: Fisher
mesh_terms: D001360:Avidin; D001710:Biotin; D002232:Carbocyanines / Q000737:chemistry*; D002467:Cell Nucleus; D003315:Cornea / Q000166:cytology; D004247:DNA / Q000032:analysis*; D005347:Fibroblasts / Q000166:cytology; D005456:Fluorescent Dyes / Q000737:chemistry*; D015870:Gene Expression; D006367:HeLa Cells; D006657:Histones / Q000235:genetics; D006735:Horseradish Peroxidase; D006801:Humans; D007124:Immunoenzyme Techniques; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007399:Interphase; D012313:RNA / Q000032:analysis*; D016196:S Phase; D014439:Tyramine / Q000737:chemistry*
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D006657:Histones; C089187:cyanine dye 3; D001360:Avidin; D012313:RNA; D001710:Biotin; D004247:DNA; D006735:Horseradish Peroxidase; D014439:Tyramine