Quantification of ANP mRNA in primary cultures of adult rat atrial myocytes by image processing: in situ hybridization to multiple parallel samples using single-stranded cDNA probes.

In primary cultures of adult rat atrial myocytes, we quantified the accumulation of atrial natriuretic peptide (ANP) mRNA in parallel with ANP secretion. ANP mRNA was quantified by image analysis of myocytes hybridized in situ with single-stranded cDNA probes generated by two successive thermal cycling procedures. In situ analysis permitted measurement of many small experimental samples in tandem while avoiding the possibility of differential extraction and processing of mRNA from sample to sample. The single-step application of 32P-labeled probes allowed processing of many parallel samples and generated intense punctate autoradiographic signals that were readily countable by image processing. Biotin-labeled probes, in conjunction with gold-labeled anti-biotin antibodies and silver intensification, gave an apparently equivalent specific signal but presented more difficulty in uniform processing of many samples and was harder to quantify by our image processing system. Measurement of ANP mRNA during atrial myocyte culture showed that ANP mRNA accumulated from undetectable levels after 1 day of culture to maximal levels by Day 8. In contrast, secretion of ANP (which is stored in atrial granules) slowly decreased, but was not abolished, during the first 5 days of culture. Subsequently, ANP secretion increased, with the increase trailing ANP mRNA accumulation by at least 24 hr.