Fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification for sensitive DNA and mRNA detection

Abstract

 We have used horseradish peroxidase-labeled 40 mer oligodeoxynucleotides (HRP-ODNs) specific for the human cytomegalovirus immediate early gene (HCMV-IE) and a novel dinitrophenol-tyramide signal amplification reagent (DNP-TSA plus) to evaluate their utility in fluorescence in situ hybridization (FISH). For DNA FISH, single or cocktails of HRP-ODNs were hybridized to metaphase chromosomes of rat 9G cells which, as determined by DNA fiber FISH, carry an integrated tandem repeat of 50–60 copies of the HCMV-IE gene. With one layer of DNP-TSA plus deposition and subsequent detection with a fluorochrome-conjugated antibody, four HRP-ODNs were needed to detect the HCMV-IE integration site. When employing two TSA amplification rounds, one HRP-ODN was sufficient for obtaining a strong signal of the integrated gene cluster, indicating that 50–60 HRP molecules can be detected with ease. In addition to DNA FISH, we report here the first use of HRP-ODN probes for mRNA detection by FISH. A single HRP-ODN and one DNP-TSA plus step resulted in clear visualization of the HCMV-IE gene transcripts in rat 9G cells induced for HCMV-IE expression by cycloheximide. Two TSA detection steps enhanced signal intensities even further. Parallel experiments with hapten-labeled ODN and cDNA probes and conventional detection methods illustrated the superiority of the HRP-ODN/TSA approach in DNA and RNA FISH.