Visualization of endoplasmic reticulum localized mRNAs in mammalian cells

J Vis Exp. 2012 Dec 17;(70):e50066. doi: 10.3791/50066.

Abstract

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • COS Cells
  • Cell Line, Tumor
  • Cell Membrane Permeability
  • Chlorocebus aethiops
  • Digitonin / pharmacology
  • Endoplasmic Reticulum / chemistry
  • Endoplasmic Reticulum / genetics*
  • Endoplasmic Reticulum / metabolism
  • Eukaryotic Cells
  • Humans
  • In Situ Hybridization, Fluorescence / methods*
  • Microscopy, Fluorescence / methods*
  • Poly A / genetics
  • RNA, Messenger / analysis*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism

Substances

  • RNA, Messenger
  • Poly A
  • Digitonin