A quantitative ‘in-situ’ hybridization method using computer-assisted image analysis. Validation and measurement of atrial-natriuretic-factor mRNA in the rat heart

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Abstract
We have validated a quantitative ‘in-situ’ hybridization method and computer-assisted image analysis of autoradiographs as a technique for measuring atrial-natriuretic-factor (ANF) mRNA in tissue sections of rat heart by: (i) producing radioactive standards to calibrate the autoradiograms and (ii) assessing: (a) specificity (through RNAase A background subtraction, comparison of ANF mRNA and non-ANF mRNA probe binding to sections, Northern analysis and section-thickness titration curves); (b) sensitivity (by calculating the limit of detection for ventricular levels of ANF mRNA); (c) precision [inter-assay CV (coefficient of variation) less than 10%; intra-assay CV 6-7%]; and (d) accuracy. We have found with this technique that deoxycortone and saline treatment of rats elevates ANF mRNA to a larger extent in the ventricles than in the atria and that, in neonatal-rat hearts, ANF mRNA is elevated in all cardiac chambers relative to adult levels.

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