DETECTION OF MESSENGER-RNA BY INSITU HYBRIDIZATION AND IN NORTHERN BLOT ANALYSIS USING OLIGODEOXYNUCLEOTIDE PROBES LABELED WITH ALKALINE-PHOSPHATASE

Aims: 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. Method: 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 also 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.