A comparative study of digoxigenin, 2,4-dinitrophenyl, and alkaline phosphatase as deoxyoligonucleotide labels in non-radioisotopic in situ hybridisation
| Autor: | J. H. Pringle, Tom Brown, A. S. J. Stewart, Elaine M. Bailey, Catherine M. McKeen, S. J. Harper, John Feehally |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
Palatine Tonsil
In situ hybridization Biology Pathology and Forensic Medicine Histones chemistry.chemical_compound Labelling Humans Digoxigenin RNA Messenger In Situ Hybridization chemistry.chemical_classification Messenger RNA Oligonucleotide General Medicine Alkaline Phosphatase Molecular biology Enzyme chemistry Biochemistry Alkaline phosphatase Dinitrophenyl 2 4-Dinitrophenol Oligonucleotide Probes Research Article |
| DOI: | 10.1136/jcp.50.8.686 |
| Popis: | AIM: To determine the optimum form of labelling and the most efficient reporter molecule for non-radioisotopic in situ hybridisation (ISH). METHODS: Nine deoxyoligonucleotides complementary to histone mRNA were synthesised and labelled either enzymatically or during solid-phase synthesis with the reporter molecules digoxigenin, 2,4-dinitrophenyl (DNP), or alkaline phosphatase. Pooled deoxyoligonucleotide cocktails were then used in non-radioisotopic ISH detection of histone mRNA in human tonsil. Hybrid detection was by nitroblue tetrazoleum/5-bromo-4-chloro-3-indolyl phosphate colorimetric development. RESULTS: The use of a spacer in 3' enzymatic labelling and when labelling with alkaline phosphatase significantly increased ISH signal. The 3' and 5' labelling of oligonucleotides with triple DNP groups during solid-phase synthesis produced the strongest signal as determined by the highest cell signal intensity and shortest development time. CONCLUSIONS: 3' and 5' solid-phase labelling with triple DNP groups produced the best labelling for non-isotopic ISH using deoxyoligonucleotide cocktails. |
| Databáze: | OpenAIRE |
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