Flow cytometric detection of beta-globin mRNA in murine haemopoietic tissues using fluorescent in situ hybridization

Abstract

The novel method for flow cytometric detection of cellular RNA species in suspended cells by fluorescent in situ hybridization (FC-FISH) was applied in the evaluation of beta-globin expression in murine haemopoietic tissues. Normal murine bone marrow cells and regenerating bone marrow cells obtained after lethal irradiation and bone marrow transplantation as well as murine 15 d fetal liver were examined. Furthermore, spleens and bone marrow of phenylhydrazine-induced anaemic mice were studied. Biotinylated sense- and antisense single strand RNA probes, obtained by transcription of a 510 nucleotides murine beta-globin cDNA sequence subcloned into the pGEM1 plasmid were used as hybridization probes. For detection of the hybrids formed, avidin-FITC was used. Only the antisense beta-globin probe gave strongly positive fluorescence signals in a defined population of cells in each of the tissues examined, whereas the sense probe did not give signals higher than control samples. Melting characteristics of the hybrids showed the specificity of the in situ hybridization reaction. Forward light scatter distributions, reflecting cell size of the positive cells were as expected from erythroid cells. Within the erythrocyte subpopulation both beta-globin-negative and -positive cells were detected. The percentages of positive cells determined flow cytometrically correlated with the percentages observed in May-Grünwald/Giemsa stained preparations. Differences observed in fluorescence intensity between positive cells of different organs were no larger than about a factor of two, indicating a rather constant beta-globin mRNA content over the entire differentiation range. An exception was 15 d fetal liver, which was shown biochemically to contain about eight times more beta-globin RNA and which had a 2.4 times higher fluorescence intensity. We estimate that the sensitivity of the present method is such that as little as 500 copies per cell of a specific mRNA of 1 kb length would be detectable.