Detection of mRNA by in situ hybridisation and in northern blot analysis using oligodeoxynucleotide probes labelled with alkaline phosphatase.
OBJECTIVE
To assess whether a reduction in intensity of signal observed using an alkaline phosphatase labelled oligodeoxynucleotide probe could be explained on the basis of procedural steps rather than reduced sensitivity.


METHODS
Signal intensity was assessed on in situ hybridisation for pro-opiomelanocortin (POMC) mRNA in rat pituitary and for somatostatin mRNA in human pancreas and in northern blot analysis for POMC mRNA in the presence and absence of formamide. The direct effects of formamide on the alkaline phosphatase detection step were assessed using histochemical enzyme detection in rat kidney.


RESULTS
All signals were reduced in systems containing formamide.


CONCLUSIONS
In the absence of formamide clear, strong signals for specific mRNAs can be obtained by in situ hybridisation and northern blot analysis using oligodeoxynucleotide probes directly labelled with alkaline phosphatase. Formamide seems to inhibit the activity of alkaline phosphatase.
University Department of Pathology, Royal Infirmary, Glasgow, Scotland.
M A
MA
Farquharson
R
R
Harvie
A
A
Kennedy
A M
AM
McNicol
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D005559:Formamides / Q000494:pharmacology; D006651:Histocytochemistry; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D007668:Kidney / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010179:Pancreas / Q000737:chemistry; D010902:Pituitary Gland / Q000737:chemistry; D011333:Pro-Opiomelanocortin / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013004:Somatostatin / Q000235:genetics
D005559:Formamides; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D013004:Somatostatin; D011333:Pro-Opiomelanocortin; D000469:Alkaline Phosphatase