| Autor: |
van de Corput MP; Department of Molecular Cell Biology, Leiden University Medical Centre., Dirks RW, van Gijlswijk RP, van de Rijke FM, Raap AK |
| Jazyk: |
angličtina |
| Zdroj: |
Histochemistry and cell biology [Histochem Cell Biol] 1998 Oct; Vol. 110 (4), pp. 431-7. |
| DOI: |
10.1007/s004180050304 |
| Abstrakt: |
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: |
MEDLINE |
| Externí odkaz: |
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