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Nucleic Acids Res. 2015 Oct 30;43(19):e126. doi: 10.1093/nar/gkv614. Epub 2015 Jun 22.

ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues.

Author information

1
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
2
Brain Science Institute, RIKEN, Hirosawa, Wako City, Saitama 351-0198, Japan JSPS Research fellow, Japan Society of Promotion of Science, 5-3-1 Kojimachi, Chiyoda-ku,Tokyo 102-0083, Japan.
3
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
4
Advanced Science Institute, RIKEN, Hirosawa, Wako City, Saitama 351-0198, Japan.
5
Advanced Science Institute, RIKEN, Hirosawa, Wako City, Saitama 351-0198, Japan Research Center for Advanced Science and Technology, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8904, Japan.
6
Brain Science Institute, RIKEN, Hirosawa, Wako City, Saitama 351-0198, Japan.
7
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan dwang@icems.kyoto-u.ac.jp.

Abstract

Elucidating the dynamic organization of nuclear RNA foci is important for understanding and manipulating these functional sites of gene expression in both physiological and pathological states. However, such studies have been difficult to establish in vivo as a result of the absence of suitable RNA imaging methods. Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks. Upon in vivo electroporation, exciton-controlled sequence-specific oligonucleotide probes revealed focally concentrated endogenous 28S rRNA and U3 snoRNA at nucleoli and poly(A) RNA at nuclear speckles. Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue. Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior.

PMID:
26101260
PMCID:
PMC4627062
DOI:
10.1093/nar/gkv614
[Indexed for MEDLINE]
Free PMC Article

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