A comparative study of digoxigenin, 2,4-dinitrophenyl, and alkaline phosphatase as deoxyoligonucleotide labels in non-radioisotopic in situ hybridisation.

Journal article

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.

Authors: Harper, S; Bailey, E; McKeen, C; Stewart, A; Pringle, J

• Publication status: Published
• Journal: Journal of clinical pathology
• Volume: 50
• Issue: 8
• Pages: 686-690
• Publication date: 1997-08-01
• DOI: 10.1136/jcp.50.8.686
• EISSN: 1472-4146
• ISSN: 0021-9746
• Language: English
• Keywords: Histones; Humans; Alkaline Phosphatase; Digoxigenin; In Situ Hybridization; Palatine Tonsil; RNA, Messenger; 2,4-Dinitrophenol; Oligonucleotide Probes