Four fluorochromes for the demonstration and microfluorometric estimation of RNA

Microfluorometric estimates of total RNA were made in selected test material stained with berberine sulfate, acridine orange, and Hoechst 33258. These measurements were compared with those obtained with propidium iodide, which is known to interact only with double-stranded nucleic acids. It was observed that all of the fluorochromes, including propidium iodide, yielded very similar patterns of fluorescence in the various types of material tested. In isolated thymocyte and hepatocyte nuclei stained with either propidium iodide or Hoechst 33258 at pH 2, it was evident that RNA could be estimated only indirectly by measuring the amount of fluorescence before and after extraction with RNase. It was apparent that the total fluorescence of small thymocyte nuclei was affected much less by RNase extraction than that of 2c hepatocyte nuclei. Attempts to obtain direct estimates of RNA by exposing the preparations to DNase were not successful: the fluorescence of thymocyte nuclei dropped almost to zero, and hepatocyte nuclei could no longer be assigned to distinct ploidy classes. In addition, since the highly condensed chromatin of thymocyte nuclei was stained much more prominently than the looser chromatin of hepatocyte nuclei with Hoechst 33258, it was apparent that this fluorochrome - when used at pH 2 - has potential usefulness as a "probe" of organizational differences in chromatin.