Fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification for sensitive DNA and mRNA detection.
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.
van de Corput
M P
MP
Department of Molecular Cell Biology, Leiden University Medical Centre.
Dirks
R W
RW
van Gijlswijk
R P
RP
van de Rijke
F M
FM
Raap
A K
AK
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis*; D004140:Dinitrophenols; D017781:Genes, Immediate-Early; D006735:Horseradish Peroxidase; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007202:Indicators and Reagents; D008677:Metaphase; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis; D051381:Rats; D016662:Virus Integration
D000956:Antigens, Viral; D004279:DNA, Viral; D004140:Dinitrophenols; D017874:Immediate-Early Proteins; D007202:Indicators and Reagents; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012367:RNA, Viral; C044582:immediate-early proteins, cytomegalovirus; D006735:Horseradish Peroxidase