Combined DNA-RNA Fluorescent <em>In situ</em> Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Tahsin Stefan Barakat; Joost Gribnau

doi:10.3791/51628

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactiva...

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Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

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15:54 min

June 14, 2014

DOI: 10.3791/51628-v

Tahsin Stefan Barakat¹, Joost Gribnau¹

¹Department of Reproduction and Development,Erasmus MC - University Medical Center

Overview

This protocol describes the simultaneous detection of RNA and DNA using fluorescent in situ hybridization (FISH) to study X chromosome inactivation in female mouse embryonic stem cells. The method allows researchers to observe nucleic acids in their native cellular environment.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Genetics

Background

X chromosome inactivation is a crucial process in female mammals.
Understanding this process can provide insights into gene regulation.
FISH is a powerful technique for visualizing nucleic acids.
This study focuses on differentiated female mouse embryonic stem cells.

Purpose of Study

To investigate X chromosome inactivation mechanisms.
To utilize FISH for simultaneous detection of RNA and DNA.
To enhance understanding of embryonic development.

Methods Used

Induction of differentiation in female mouse embryonic stem cells.
Fixation of cells using paraldehyde.
Preparation of probes for detecting DNA and RNA.
Labeling DNA fragments with biotin or digoxigenin.

Main Results

Successful simultaneous detection of RNA and DNA.
Observation of enhanced transcription during X chromosome inactivation.
Accumulation of cyst RNA coding for the inactive X chromosome.
Validation of the FISH technique for studying gene regulation.

Conclusions

The protocol effectively demonstrates X chromosome inactivation.
FISH is a valuable tool for studying nucleic acids in situ.
This method can be applied to other genetic studies.

Frequently Asked Questions

What is FISH?

FISH stands for fluorescent in situ hybridization, a technique used to detect specific nucleic acid sequences in cells.

Why study X chromosome inactivation?

Studying X chromosome inactivation helps understand gene regulation and developmental processes in female mammals.

What are the key steps in the protocol?

Key steps include inducing differentiation, fixing cells, preparing probes, and performing FISH.

How does this method contribute to neuroscience?

This method provides insights into genetic regulation that can affect neural development and function.

Can this technique be used for other types of cells?

Yes, FISH can be adapted for use in various cell types to study different genetic phenomena.

What is the significance of cyst RNA?

Cyst RNA is associated with the inactive X chromosome and plays a role in X chromosome inactivation.

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

The overall goal of this procedure is to study X chromosome inactivation in differentiated female mouse embryonic stem cells by means of simultaneously combined DNA and RNA fluorescent in C two hybridization or D-N-A-R-N-A fish. This is accomplished by first inducing differentiation in female mouse embryonic stem cells. As during this process, which simulates normal post implantation embryonic development, X chromosome inactivation is initiated, resulting in enhanced transcription and accumulation of cyst RNA, coding the future inactive X chromosome.

The second step is to fix the differentiated embryonic stem cells using paraldehyde. Next, prepare the probes for detecting both DNA from the X chromosome and cyst RNA by labeling DNA fragments with biotin or digo genin. Using Nick translation.

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