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

Oomoto I, Suzuki-Hirano A, Umeshima H, Han YW, Yanagisawa H, Carlton P, Harada Y, Kengaku M, Okamoto A, Shimogori T, Wang DO - Nucleic Acids Res. (2015)

Bottom Line: Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

No MeSH data available.


Related in: MedlinePlus

ECHO-liveFISH imaging did not alter the expression level or the function of the target RNA. (A) Total RNA was isolated from transfected HeLa cells and specific target RNAs were quantified with RT-qPCR. The graphs show the expression levels of U3 snoRNA, 28S rRNA, 18S rRNA and GAPDH (normalized to ‘D514-random’-transfected cells). Note that no significant difference was induced by transfection of target-specific ECHO probes (one-way ANOVA). (B) Estimation of global translational activities in the probe-transfected HeLa cells with puromycin pulse-labeling and immunoblotting (27). Immunodensitometry in each individual lane was analyzed and normalized to that of the ‘D514-random’ lane. CHX, cycloheximide, a translational inhibitor. (C) Left, Confocal images (FV1000) of ECHO probe-transfected HeLa cells immunostained with a puromycin-specific antibody (magenta) and DAPI (cyan). Cellular distribution of newly synthesized proteins was unaffected among cell groups. Right, Quantification of mean fluorescence staining intensity (MFI) of puromycin showed undetectable changes in the gross translational activity among cell groups. A.U.: arbitrary units. Scale bar: 20 μm. DAPI: 405 nm excitation, 425–475 nm detection; Alexa 633: 635 nm excitation, 650–750 nm detection; 0.165 × 0.165 μm pixel size; 20 μs pixel dwell times.
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Figure 3: ECHO-liveFISH imaging did not alter the expression level or the function of the target RNA. (A) Total RNA was isolated from transfected HeLa cells and specific target RNAs were quantified with RT-qPCR. The graphs show the expression levels of U3 snoRNA, 28S rRNA, 18S rRNA and GAPDH (normalized to ‘D514-random’-transfected cells). Note that no significant difference was induced by transfection of target-specific ECHO probes (one-way ANOVA). (B) Estimation of global translational activities in the probe-transfected HeLa cells with puromycin pulse-labeling and immunoblotting (27). Immunodensitometry in each individual lane was analyzed and normalized to that of the ‘D514-random’ lane. CHX, cycloheximide, a translational inhibitor. (C) Left, Confocal images (FV1000) of ECHO probe-transfected HeLa cells immunostained with a puromycin-specific antibody (magenta) and DAPI (cyan). Cellular distribution of newly synthesized proteins was unaffected among cell groups. Right, Quantification of mean fluorescence staining intensity (MFI) of puromycin showed undetectable changes in the gross translational activity among cell groups. A.U.: arbitrary units. Scale bar: 20 μm. DAPI: 405 nm excitation, 425–475 nm detection; Alexa 633: 635 nm excitation, 650–750 nm detection; 0.165 × 0.165 μm pixel size; 20 μs pixel dwell times.

Mentions: Statistical analysis were done with Excel (Microsoft), Graphpad Prism (Graphpad software) and EZR (32). The P-values associated with comparisons in Figure 3 were based on One-way ANOVA and the P-values associated with other comparisons were based on Student's t-test. Results are plotted as mean ± SEM from three independent experiments, unless otherwise indicated in the figure legends.


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

Oomoto I, Suzuki-Hirano A, Umeshima H, Han YW, Yanagisawa H, Carlton P, Harada Y, Kengaku M, Okamoto A, Shimogori T, Wang DO - Nucleic Acids Res. (2015)

ECHO-liveFISH imaging did not alter the expression level or the function of the target RNA. (A) Total RNA was isolated from transfected HeLa cells and specific target RNAs were quantified with RT-qPCR. The graphs show the expression levels of U3 snoRNA, 28S rRNA, 18S rRNA and GAPDH (normalized to ‘D514-random’-transfected cells). Note that no significant difference was induced by transfection of target-specific ECHO probes (one-way ANOVA). (B) Estimation of global translational activities in the probe-transfected HeLa cells with puromycin pulse-labeling and immunoblotting (27). Immunodensitometry in each individual lane was analyzed and normalized to that of the ‘D514-random’ lane. CHX, cycloheximide, a translational inhibitor. (C) Left, Confocal images (FV1000) of ECHO probe-transfected HeLa cells immunostained with a puromycin-specific antibody (magenta) and DAPI (cyan). Cellular distribution of newly synthesized proteins was unaffected among cell groups. Right, Quantification of mean fluorescence staining intensity (MFI) of puromycin showed undetectable changes in the gross translational activity among cell groups. A.U.: arbitrary units. Scale bar: 20 μm. DAPI: 405 nm excitation, 425–475 nm detection; Alexa 633: 635 nm excitation, 650–750 nm detection; 0.165 × 0.165 μm pixel size; 20 μs pixel dwell times.
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Related In: Results  -  Collection

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Figure 3: ECHO-liveFISH imaging did not alter the expression level or the function of the target RNA. (A) Total RNA was isolated from transfected HeLa cells and specific target RNAs were quantified with RT-qPCR. The graphs show the expression levels of U3 snoRNA, 28S rRNA, 18S rRNA and GAPDH (normalized to ‘D514-random’-transfected cells). Note that no significant difference was induced by transfection of target-specific ECHO probes (one-way ANOVA). (B) Estimation of global translational activities in the probe-transfected HeLa cells with puromycin pulse-labeling and immunoblotting (27). Immunodensitometry in each individual lane was analyzed and normalized to that of the ‘D514-random’ lane. CHX, cycloheximide, a translational inhibitor. (C) Left, Confocal images (FV1000) of ECHO probe-transfected HeLa cells immunostained with a puromycin-specific antibody (magenta) and DAPI (cyan). Cellular distribution of newly synthesized proteins was unaffected among cell groups. Right, Quantification of mean fluorescence staining intensity (MFI) of puromycin showed undetectable changes in the gross translational activity among cell groups. A.U.: arbitrary units. Scale bar: 20 μm. DAPI: 405 nm excitation, 425–475 nm detection; Alexa 633: 635 nm excitation, 650–750 nm detection; 0.165 × 0.165 μm pixel size; 20 μs pixel dwell times.
Mentions: Statistical analysis were done with Excel (Microsoft), Graphpad Prism (Graphpad software) and EZR (32). The P-values associated with comparisons in Figure 3 were based on One-way ANOVA and the P-values associated with other comparisons were based on Student's t-test. Results are plotted as mean ± SEM from three independent experiments, unless otherwise indicated in the figure legends.

Bottom Line: Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

No MeSH data available.


Related in: MedlinePlus