Sensitive detection of RNAs in single cells by flow cytometry.
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.
Yu
H
H
Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, PA 15213.
Ernst
L
L
Wagner
M
M
Waggoner
A
A
D001483:Base Sequence; D015342:DNA Probes / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D006367:HeLa Cells; D006657:Histones / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis*; D012337:RNA, Ribosomal, 18S / Q000032:analysis* / Q000235:genetics; D012339:RNA, Ribosomal, 28S / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity
D015342:DNA Probes; D006657:Histones; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S