Related Experiment Video

Updated: May 15, 2026

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells
10:24

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

Published on: December 17, 2012

Visualization of endoplasmic reticulum localized mRNAs in mammalian cells

Xianying A Cui1, Alexander F Palazzo

  • 1Department of Biochemistry, University of Toronto.

Journal of Visualized Experiments : Jove
|December 29, 2012
PubMed

Insights

Researchers developed a new method to visualize messenger RNAs (mRNAs) at the endoplasmic reticulum (ER) surface. This technique allows for clear observation and quantification of ER-associated mRNAs, aiding in understanding mRNA-ER interactions.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Most eukaryotic messenger RNAs (mRNAs) encoding secreted and membrane proteins associate with the endoplasmic reticulum (ER).
  • Visualizing ER-bound mRNAs is challenging due to the presence of non-targeted (
  • free,') transcripts obscuring the view.
  • Understanding mRNA localization to the ER is crucial for protein synthesis and cellular function.

Purpose of the Study:

  • To develop a reliable method for visualizing and quantifying ER-associated mRNAs.
  • To overcome the limitations of existing techniques in distinguishing ER-bound from free mRNAs.
  • To facilitate the investigation of mRNA-ER interactions.

Main Methods:

  • Developed a digitonin-based selective permeabilization protocol to remove cytoplasmic contents while preserving ER integrity.
  • Combined this extraction method with fluorescent in situ hybridization (FISH).
  • Utilized fluorescent microscopy for visualization and quantification of ER-bound mRNAs.

Main Results:

  • The digitonin-FISH method successfully visualizes ER-bound mRNAs with high clarity.
  • The protocol allows for the assessment and quantification of ER-association for both bulk poly(A) transcripts and specific mRNAs.
  • The technique effectively distinguishes ER-associated mRNAs from free cytoplasmic transcripts.

Conclusions:

  • The developed digitonin-FISH method provides a robust approach to study mRNA localization at the ER.
  • This technique enables quantitative analysis of mRNA-ER association.
  • The assay is valuable for exploring the mechanisms underlying mRNA-ER interactions in eukaryotic cells.

More Related Videos

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells16:43

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

Published on: February 18, 2014

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers07:39

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Published on: June 4, 2019

Related Concept Videos

Endoplasmic Reticulum01:39

Endoplasmic Reticulum

The Endoplasmic Reticulum (ER) in eukaryotic cells is a substantial network of interconnected membranes with diverse functions, from calcium storage to biomolecule synthesis. A primary component of the endomembrane system, the ER manufactures phospholipids critical for membrane function throughout the cell. Additionally, the two distinct regions of the ER specialize in the manufacture of specific lipids and proteins.
The Endoplasmic Reticulum01:43

The Endoplasmic Reticulum

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary protein and lipid synthesis factory for most cell organelles, such as the Golgi apparatus, lysosomes, secretory vesicles, and the plasma membrane. Despite being the most extensive and functionally complex subcellular organelle, ER was the last to be discovered. After years of deliberation, Keith Porter and George Palade in the year 1954,...
The Endoplasmic Reticulum01:43

The Endoplasmic Reticulum

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary protein and lipid synthesis factory for most cell organelles, such as the Golgi apparatus, lysosomes, secretory vesicles, and the plasma membrane. Despite being the most extensive and functionally complex subcellular organelle, ER was the last to be discovered. After years of deliberation, Keith Porter and George Palade in the year 1954,...
Smooth Endoplasmic Reticulum01:21

Smooth Endoplasmic Reticulum

Smooth endoplasmic reticulum or smooth ER is a sub-organelle with specialized functions in animal cells and plant cells. It is often associated with the tubule morphology of the endoplasmic reticulum.
The ER provides optimal conditions for synthesizing steroid hormones and lipids, such as phospholipids and triglycerides. Traditionally, lipid metabolism was considered to be a smooth ER function. However, there is no direct evidence to prove that rough ER is completely excluded from lipid...
Golgi Apparatus01:49

Golgi Apparatus

As they leave the Endoplasmic Reticulum (ER), properly folded and assembled proteins are selectively packaged into vesicles. These vesicles are transported by microtubule-based motor proteins and fuse together to form vesicular tubular clusters, subsequently arriving at the Golgi apparatus, a eukaryotic endomembrane organelle that often has a distinctive ribbon-like appearance.
Post-translational Translocation of Proteins to the RER01:27

Post-translational Translocation of Proteins to the RER

A sizable fraction of proteins destined for ER are first synthesized in the cell cytosol and then transported across the ER membrane–a process called post-translational translocation. Similar to cotranslationally translocated proteins, these proteins also use the Sec translocon complex to enter the ER lumen.
Targeting proteins to the ER
Hsp40 and Hsp70 chaperone molecules bind the translated proteins in the cytosol to prevent their folding. The chaperone binding helps to keep the signal...