Application of biotinylated oligonucleotide probes to the detection of pituitary hormone mRNA using Northern blot analysis, in situ hybridization at the light- and electron-microscope levels

Abstract

There have been many reports on radioisotopic in situ hybridization (ISH) studies for the demonstration of pituitary hormone mRNAs in normal pituitary gland and pituitary adenomas. Recent studies have revealed that non-radioisotopic ISH has several advantages over the radioisotopic method. Using ISH with biotinylated oligonucleotide probes, we have been able to localize various pituitary hormone mRNAs in paraffin wax or frozen sections of rat normal pituitary gland and human pituitary adenomas. For control studies we used ISH with sense probes, ISH without probes, pretreatment with ribonuclease, ISH with a probe for beta-actin and Northern blot hybridization. Using biotinylated probes, gene transcripts of rat growth hormone and prolactin were detected by Northern blot hybridization. The same biotinylated probes were used not for light microscope ISH but also for the electron microscopical demonstration of rat growth hormone and prolactin mRNAs on the polysomes of the rough endoplasmic reticula. It is emphasized that biotinylated oligonucleotide probes are useful for the analysis of pituitary endocrine function because they are applicable to the three hybridization methods, namely, Northern blot hybridization and ISH at the light and electron microscope levels.