Visualizing nucleic acids in living cells by fluorescence in vivo hybridization.
The analysis of the spatial-dynamic properties of DNA and RNA molecules in living cells will greatly extend our knowledge of genome organization and gene expression regulation in the cell nucleus. The development of hybridization methods allowing detection of specific endogenous DNA and RNA sequences in living cells has therefore been a challenge for many years. However, there are many technical issues that have proven so far to be difficult, or even impossible, to overcome. As a result, in most situations, the application of in vivo hybridization methods is currently limited to the visualization of highly repetitive DNA sequences or abundant RNA species. We describe a protocol that enables the visualization and tracking of telomeres in living cells by hybridization with a fluorescent peptide nucleic acid (PNA) probe. Furthermore, we describe a method that allows the detection of abundant endogenous RNAs in living cells by microinjecting fluorescently labeled complementary 2'-O-methyl RNA probes.
Wiegant
Joop
J
Department of Molecular Cell Biology, Leiden University Medical Centre, Leiden, The Netherlands.
Brouwer
Anneke K
AK
Tanke
Hans J
HJ
Dirks
Roeland W
RW
D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D004247:DNA / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008845:Microinjections; D057054:Molecular Imaging / Q000379:methods*; D020135:Peptide Nucleic Acids / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*
D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids; D012313:RNA; D004247:DNA