Fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification for sensitive DNA and mRNA detection
| Autor: | Frans M. van de Rijke, Mariëtte P.C. van de Corput, A. K. Raap, Rob P.M. van Gijlswijk, Roeland W. Dirks |
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| Rok vydání: | 1998 |
| Předmět: |
Histology
Virus Integration viruses Cytomegalovirus In situ hybridization Horseradish peroxidase Cell Line Immediate-Early Proteins chemistry.chemical_compound Tandem repeat Complementary DNA Gene cluster medicine Animals Humans RNA Messenger Cloning Molecular Antigens Viral Genes Immediate-Early Molecular Biology Gene Horseradish Peroxidase In Situ Hybridization Fluorescence Metaphase medicine.diagnostic_test biology hemic and immune systems Cell Biology biochemical phenomena metabolism and nutrition respiratory system Molecular biology Rats Medical Laboratory Technology chemistry DNA Viral biology.protein RNA Viral Indicators and Reagents Oligonucleotide Probes Dinitrophenols DNA Fluorescence in situ hybridization |
| Zdroj: | Histochemistry and Cell Biology. 110:431-437 |
| ISSN: | 1432-119X 0948-6143 |
| DOI: | 10.1007/s004180050304 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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