Abstract

The detection of small amounts of biologically active material in animals may require quantification of radioactive label. Here we compare several image analysis methods for quantifying relative levels of radioactive probe detected by x-ray film or photographic emulsion. Total RNA "dot blots" and ovine muscle sections were probed for the presence of muscle actin mRNA using 35S-labelled riboprobes. Bound probe was detected by x-ray film in both cases. The probed sections were further exposed to photographic emulsion, while the dot blots were individually quantified by scintillation counting. Bound probe was quantified macroscopically by digitisation of optical density (OD) and thresholding of the x-ray film image. Dot blot image ODs were correlated with scintillation counts (r>0.96), demonstrating that the levels of mRNA detected in tissues by autoradiography are proportional to cellular mRNA concentration. Densitometry of section macroautoradiographic images can therefore be useful in determining relative probe signal at the tissue level. When probe was quantified microscopically by counting silver grains in emulsion autoradiographs, simple thresholding correlated with manual counts (r=0.88) but was unreliable at higher grain densities. Processing of the gray image gave a more direct estimate of silver grain density (r= 0.98).

RG, Sherlock, HT Blair, B Kuhn, DDS MacKenzie, BH Breier, PD Gluckman, and SN McCutcheon

Proceedings of the New Zealand Society of Animal Production, Volume 53, , 381-384, 1993