Visualizing RNA molecules inside the nucleus of living cells.
Fluorescence in situ hybridization is a widely used technique in cell biology providing insight into the spatial organization of specific RNA transcripts in the cell nucleus. However, to further investigate the dynamics of the transcription process and the transport rates of RNAs through the nucleus, RNAs need to be visualized and tracked in the living cell. In past years, various methods have been developed with the aim of tagging specific RNAs with a fluorescent moiety without interfering with cell vitality. These methods include the delivery of probes into a living cell, the in vivo hybridization of fluorescent oligonucleotide probes to endogenous RNAs, and the microscopic imaging of the tagged RNAs in living cells. In this article, we review a number of methods for tagging and visualizing endogenous RNAs in living cells. In addition, a protocol is described that allows detection of various RNA types using fluorochrome-labeled 2(')-O-methyl oligoribonucleotide (2(')-OMe RNA) probes. Compared with conventional oligodeoxynucleotide probes, 2(')-OMe RNA probes are not degraded by nucleases, form stable hybrids with structured RNAs, and do not interfere with cell vitality.
Dirks
Roeland W
RW
Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL, Leiden, The Netherlands. r.w.dirks@lumc.nl
Molenaar
Chris
C
Tanke
Hans J
HJ
D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D012313:RNA / Q000032:analysis*
D012313:RNA