A comparative study of digoxigenin, 2, 4-dinitrophenyl, and alkaline phosphatase as deoxyoligonucleotide labels in non-radioisotopic in situ hybridisation. Issue 8 (August 1997)

• Record Type: Journal Article
• Authors: Harper, S J; Bailey, E; McKeen, C M; Stewart, A S; Pringle, J H; Feehally, J; Brown, T
• Is Part Of: Journal of clinical pathology. Volume 50:Issue 8(1997)
• Page Start: 686
• Page End: 690
• Publication Date: 1997-08
• Subjects: Pathology -- Periodicals; Pathology, Molecular -- Periodicals; 616.0705
• DOI: 10.1136/jcp.50.8.686
• Languages: English
• ISSNs: 0021-9746

Abstract

Abstract : 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.