A quantitative 'in-situ' hybridization method using computer-assisted image analysis. Validation and measurement of atrial-natriuretic-factor mRNA in the rat heart.
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.
Nunez
D J
DJ
Clinical Pharmacology Unit, Addenbrooke's Hospital, Cambridge, U.K.
Davenport
A P
AP
Emson
P C
PC
Brown
M J
MJ
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000235:genetics*; D001345:Autoradiography; D015152:Blotting, Northern; D007091:Image Processing, Computer-Assisted; D008297:Male; D008901:Mineralocorticoids / Q000494:pharmacology; D009206:Myocardium / Q000032:analysis*; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000032:analysis; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D012260:Ribonucleases; D012965:Sodium Chloride / Q000494:pharmacology
D008901:Mineralocorticoids; D015347:RNA Probes; D012333:RNA, Messenger; C472610:atrial natriuretic peptide, rat; D012965:Sodium Chloride; D012313:RNA; D009320:Atrial Natriuretic Factor; D012260:Ribonucleases