Journal
METHODS
Volume 29, Issue 1, Pages 51-57Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S1046-2023(02)00290-6
Keywords
in vivo hybridization; living cell; 2 '-O-methyl RNA; molecular beacon; oligodeoxynucleotides; peptide nucleic acid; fluorescence resonance energy transfer; microinjection; live cell imaging; RNA fluorescence in situ hybridization
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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. (C) 2002 Elsevier Science (USA). All rights reserved.
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