DETECTION OF INSULIN MESSENGER-RNA BY IN-SITU AND NORTHERN BLOT HYBRIDIZATION USING ISOTOPIC AND NONISOTOPIC PROBES

Northern blot hybridization was carried out to detect insulin mRNA in a variety of tissues of Wistar rats using isotopic cDNA or 17-mer oligonucleotide probes. The insert of pCRI-354, containing rat insulin I cDNA, was labelled by the random primer method using alpha-[P-32]-dATP. The result gave a single transcript of 500-650 nucleotides. Oligonucleotide probes, on the other hand, showed no specific hybridization signal in association with a high background. In situ hybridization was carried out to localize insulin mRNA in the pancreas of Wistar rats using isotopic or non-isotopic cDNA probes. The insert of pCRI-354 was labelled by the same method using either alpha-P-32-dATP or biotin-11-dUTP. Hyridization was performed under two different conditions, i.e., either moderate or low stringency. Autoradiography was performed for isotopic probes, while streptavidin alkaline phosphatase followed by a NBT-BCIP reaction was applied for biotinylated probes. Results indicated that both isotopic and biotinylated cDNA probes showed a specific signal over beta-cells of pancreas islets. The more stringent hybridization condition gave a reduced signal with less background compared to the moderate one. In situ hybridization using oligonucleotide probes gave somewhat an ambiguous result where probes appeared to hybridize non-specifically to cytoplasmic RNA.