Fluorescent in‐situ hybridization to detect cellular RNA by flow cytometry and confocal microscopy†

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Abstract
SUMMARY: A fluorescent in‐situ hybridization procedure was developed which is suitable for detection of specific cellular RNA in cells fixed in suspension. The procedure was originally developed for analysis of single‐cell suspensions by flow cytometry. The resulting fluorescent cells proved to have their 3‐D morphology prefectly preserved. The spatial distribution of specific ribosomal RNA and messenger RNA could then be analysed by confocal microscopy of individual cells. In the hybridization procedure, biotinylated single‐stranded RNA probes were used that were produced by transcription from cloned DNA fragments. Detection of poly‐(A)+ RNA was performed with a poly‐biotin‐d(U)‐tailed oligo‐d(T) probe. Bound probe was detected using streptavidin–fluorescein isothiocyanate. For flow cytometry, nuclear DNA was counterstained with di‐amidine‐2‐phenyl indol. Multi‐parameter flow cytometry was used to quantify the fluorescence intensity, i.e. the hybridization signal, of thousands of cells. Confocal scanning microscopy on individual cells revealed the intracellular distribution of the target RNA. The distribution of ribosomal RNA and poly‐(A)+ RNA mRNA in mouse bone marrow and the human leukaemia cell line HL60 cells was investigated. Ribosomal RNA was confined to the cytoplasm of the cells, although in many cells the nucleoli could also be distinguished. Poly‐(A)+ RNA in HL60 cells was found both in the cytoplasm and nucleus. A granular cytoplasmic distribution was seen in part of these cells.