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

Author:

Nunez D J1,Davenport A P1,Emson P C2,Brown M J1

Affiliation:

1. Clinical Pharmacology Unit, Addenbrooke's Hospital, Cambridge CB2 2QQ, U.K.

2. MRC Group, Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, U.K.

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.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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