QUANTIFICATION OF ANP MESSENGER-RNA IN PRIMARY CULTURES OF ADULT-RAT ATRIAL MYOCYTES BY IMAGE-PROCESSING - IN-SITU HYBRIDIZATION TO MULTIPLE PARALLEL SAMPLES USING SINGLE-STRANDED CDNA PROBES
L. Kordylewski et al., QUANTIFICATION OF ANP MESSENGER-RNA IN PRIMARY CULTURES OF ADULT-RAT ATRIAL MYOCYTES BY IMAGE-PROCESSING - IN-SITU HYBRIDIZATION TO MULTIPLE PARALLEL SAMPLES USING SINGLE-STRANDED CDNA PROBES, The Journal of histochemistry and cytochemistry, 43(5), 1995, pp. 481-488
In primary cultures of adult rat atrial myocytes, we quantified the ac
cumulation of atrial natriuretic peptide (ANP) mRNA in parallel with A
NP secretion. ANP mRNA was quantified by image analysis of myocytes hy
bridized in situ with single-stranded cDNA probes generated by two suc
cessive thermal cycling procedures. In situ analysis permitted measure
ment of many small experimental samples in tandem chile avoiding the p
ossibility of differential extraction and processing of mRNA from samp
le to sample. The single-step application of P-32-labeled probes allow
ed processing of many parallel samples and generated intense punctate
autoradiographic signals that were readily countable by image processi
ng. Biotin-labeled probes, in conjunction with gold-labeled anti-bioti
n antibodies and silver intensification, gave an apparently equivalent
specific signal bur presented more difficulty in uniform processing o
f many samples and was harder to quantify by our image processing syst
em. Measurement of ANP mRNA during atrial myocyte culture showed that
ANP mRNA accumulated from undetectable levels after 1 day of culture t
o maximal levels by Day 8. In contrast, secretion of ANP (which is sto
red in atrial granules) slowly decreased, bur was not abolished, durin
g the first 5 days of culture. Subsequently, ANP secretion increased,
with the increase trailing ANP mRNA accumulation by at least 24 hr.