Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Ragini  M. Mistry; Pankaj  K. Singh; Maureen  G. Mancini; Fabio Stossi; Michael  A. Mancini

doi:10.3791/61680

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

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Biochemistry

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JoVE Journal Biochemistry

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

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06:26 min

September 20, 2020

DOI: 10.3791/61680-v

Ragini M. Mistry^1,2, Pankaj K. Singh^1,3, Maureen G. Mancini^1,2, Fabio Stossi^1,2, Michael A. Mancini^1,2,3,4

¹GCC Center for Advanced Microscopy and Image Informatics, ²Department of Molecular and Cellular Biology,Baylor College of Medicine, ³Center for Translational Cancer Research, Institute of Biosciences and Technology,Texas A&M University, ⁴Department of Pharmacology and Chemical Biology,Baylor College of Medicine

Overview

Single molecule RNA fluorescence in situ hybridization (smFISH) is a powerful technique for quantifying RNA levels and localization at the single cell level. This method enables the analysis of cell-to-cell and allele-by-allele variation in RNA, providing insights into gene transcription and RNA response to external stimuli.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Molecular Biology

Background

smFISH allows for the investigation of RNA content in individual cells.
It provides spatial information about RNA distribution.
This method is crucial for understanding gene expression dynamics.
It can reveal variations in RNA levels due to external factors.

Purpose of Study

To validate lab protocols for smFISH.
To enable accurate quantification of specific RNAs.
To facilitate imaging and analysis of RNA at the single cell level.

Methods Used

Cells are treated with an estrogen receptor agonist.
Cells are fixed using a fixation buffer.
Washing steps are performed with cold sterile PBS.
Cells are incubated in 70% ethanol for preservation.

Main Results

smFISH accurately quantifies RNA levels.
It reveals spatial localization of specific RNAs.
Variations in RNA content can be analyzed effectively.
Results provide insights into cellular responses to stimuli.

Conclusions

smFISH is a validated method for single cell RNA analysis.
It enhances understanding of gene transcription processes.
This technique is valuable for studying RNA dynamics in response to external factors.

Frequently Asked Questions

What is smFISH?

smFISH is a technique used to quantify RNA levels and localization at the single cell level.

How does smFISH work?

It uses fluorescent probes to detect specific RNA molecules within cells.

What are the applications of smFISH?

It can be used to study gene expression, RNA localization, and cellular responses to stimuli.

What are the key steps in the smFISH protocol?

Key steps include cell fixation, washing, and incubation with fluorescent probes.

Why is single cell analysis important?

It allows for the understanding of heterogeneity in cellular responses and gene expression.

Can smFISH be used for different types of RNA?

Yes, smFISH can be tailored to detect various RNA species.

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

SM FISH accurately quantifies levels and localization of specific RNase. This method is particularly useful for analyzing cell to cell and allele by allele variation in RNA. Gene transcription is an essential process in cell biology, SM FISH allows for the investigation of RNA content in response to external stimuli at the single cell level and provide spatial information.

Following estrogen receptor agonist treatment, wash the cells with cold sterile PBS with calcium and magnesium and fix the cells with 500 microliters of fixation buffer for 30 minutes on ice. At the end of the incubation, wash the cells two times with cold sterile PBS with calcium and magnesium for three minutes before adding 500 microliters of 70%ethanol to each well. Then seal the 24 well plate with a paraffin plastic film and place the plate on a rotator at four degrees Celsius for at least four and up to 16 hours.

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