Quick Fluorescent <em>In Situ</em> Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Minghui Yue; John Lalith Charles Richard; Norishige Yamada; Akiyo Ogawa; Yuya Ogawa

doi:10.3791/52053

Veloce fluorescente In Situ Protocollo di ibridazione per Xist RNA combinata con immunof...

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Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Veloce fluorescente In Situ Protocollo di ibridazione per Xist RNA combinata con immunofluorescenza di istone modifica in cromosoma X inattivazione

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12:42 min

November 26, 2014

DOI: 10.3791/52053-v

Minghui Yue*^1,2, John Lalith Charles Richard*^1,2, Norishige Yamada^1,2, Akiyo Ogawa¹, Yuya Ogawa^1,2

¹Division of Reproductive Sciences,Cincinnati Children's Hospital Medical Center, ²Department of Pediatrics,University of Cincinnati College of Medicine

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Overview

This article presents a customizable strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. It enables the examination of RNA dynamics alongside chromatin structure and transcriptional regulation at the single-cell level.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Epigenetics

Background

Long non-coding RNAs play a crucial role in gene regulation.
Understanding RNA dynamics is essential for insights into cellular processes.
X chromosome inactivation is a key area of study in epigenetics.
Combining FISH with immunofluorescence enhances visualization of RNA and chromatin.

Purpose of Study

To investigate the dynamics of long non-coding RNAs during X chromosome inactivation.
To explore associated epigenetic modifications in differentiating embryonic stem cells.
To utilize a rapid and effective FISH protocol for detailed analysis.

Methods Used

Preparation of slides from differentiating embryonic body cells.
Permeabilization and fixation of cells for staining.
Use of antibodies to localize histone modifications on the inactive X chromosome.
Application of oligonucleotide probes for FISH to visualize RNA dynamics.

Main Results

Successful localization of long non-coding RNAs and histone modifications.
Fluorescence microscopy effectively demonstrated RNA cloud dynamics.
Insights into transcriptional regulation during embryonic stem cell differentiation.
Validation of the combined FISH and immunofluorescence approach.

Conclusions

The developed protocol is a valuable tool for studying RNA dynamics.
It provides insights into the interplay between RNA and chromatin structure.
This method can be adapted for various research applications in cell biology.

Frequently Asked Questions

What is the significance of long non-coding RNAs?

Long non-coding RNAs are crucial for regulating gene expression and play important roles in various cellular processes.

How does the FISH protocol enhance RNA visualization?

The FISH protocol allows for the specific labeling of RNA molecules, enabling detailed visualization in the context of cellular structures.

What are the applications of this study's findings?

The findings can be applied to understand gene regulation mechanisms and the role of epigenetics in development.

Can this protocol be used for other types of cells?

Yes, the protocol can be adapted for various cell types to study RNA dynamics and chromatin interactions.

What microscopy techniques are used in this study?

Fluorescence microscopy is used to visualize the localization of RNA and histone modifications.

Is this method suitable for high-throughput studies?

The protocol can be optimized for high-throughput applications, depending on the specific experimental design.

Abbiamo sviluppato un protocollo di ibridazione fluorescente in situ (FISH) facilmente personalizzabile e specifico per il filamento combinato con l'immunofluorescenza. Ciò consente un esame dettagliato della dinamica dell'RNA con una visione simultanea della struttura della cromatina, dell'organizzazione nucleare e della regolazione trascrizionale a livello di singola cellula.

L'obiettivo generale di questa procedura è quello di studiare le dinamiche dell'RNA e della modificazione epigenetica ad esso associata durante l'inattivazione del cromosoma X utilizzando una rapida ibridazione fluorescente in situ combinata con l'immunofluorescenza. Questo protocollo richiede la preparazione di vetrini con vetrini di embrione differenzianti, che vengono permeati e quindi fissati per la colorazione. Successivamente, il marcatore fluorescente localizzato da un anticorpo contro l'istone H 3 trimetillisina 27 si accumula sul cromosoma X inattivo.

La fase finale utilizza sonde oligonucleotidiche che contengono una modifica di cinque ammino prime e sono marcate con un fluoroforo reattivo medio AM per eseguire l'esistenza. Pesce a RNA. In definitiva, la microscopia a fluorescenza viene utilizzata per mostrare la localizzazione della nuvola di RNA esistente e la modifica degli istoni durante la differenziazione delle cellule E-E.

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