Yu H, Ernst L, Wagner M, Waggoner A
Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, PA 15213.
Nucleic Acids Res. 1992 Jan 11;20(1):83-8. doi: 10.1093/nar/20.1.83.
A rapid and sensitive fluorescent in situ hybridization method has been developed to probe RNA contents of individual cells by flow cytometry. Fixed cells in suspension were hybridized with 5' end-fluorophore-labeled oligodeoxynucleotides complementary to defined regions of the RNA of interest and analyzed by flow cytometry. With this method, we monitored combinations of histone H4 mRNA, 18S rRNA and 28S rRNA levels in synchronized HeLa S3 cells by multicolor analysis. A fluorescence signal equivalent to 1800 copies of histone H4 mRNA per cell was detected with signal-to-background ratio of 5.4. If non-specific binding of the fluorophore-labeled probe can be reduced, as few as 100 copies of mRNA of the size of H4 could be detected in individual cells by flow cytometry.
已开发出一种快速灵敏的荧光原位杂交方法，通过流式细胞术探测单个细胞的RNA含量。将悬浮的固定细胞与5'端荧光团标记的寡脱氧核苷酸杂交，该寡脱氧核苷酸与感兴趣RNA的特定区域互补，然后通过流式细胞术进行分析。利用这种方法，我们通过多色分析监测了同步化的HeLa S3细胞中组蛋白H4 mRNA、18S rRNA和28S rRNA水平的组合。检测到每个细胞相当于1800个组蛋白H4 mRNA拷贝的荧光信号，信号与背景的比率为5.4。如果能减少荧光团标记探针的非特异性结合，通过流式细胞术在单个细胞中可检测到低至100个H4大小的mRNA拷贝。
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