A comparative study of digoxigenin, 2,4-dinitrophenyl, and alkaline phosphatase as deoxyoligonucleotide labels in non-radioisotopic in situ hybridisation.
OBJECTIVE
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.
Harper
S J
SJ
Richard Bright Renal Unit, Southmead Hospital, Westbury-on-Trym, Bristol, UK.
Bailey
E
E
McKeen
C M
CM
Stewart
A S
AS
Pringle
J H
JH
Feehally
J
J
Brown
T
T
D019297:2,4-Dinitrophenol; D000469:Alkaline Phosphatase; D004076:Digoxigenin; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D014066:Palatine Tonsil / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics
D006657:Histones; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D000469:Alkaline Phosphatase; D004076:Digoxigenin; D019297:2,4-Dinitrophenol