Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

Xianying A. Cui; Alexander F. Palazzo

doi:10.3791/50066

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

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Biology

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JoVE Journal Biology

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

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10:24 min

December 17, 2012

DOI: 10.3791/50066-v

Xianying A. Cui¹, Alexander F. Palazzo¹

¹Department of Biochemistry,University of Toronto

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

The aim of this procedure is to visualize messenger RNA that is associated with the endoplasmic reticulum or er. This is accomplished by first plating mammalian tissue culture cells on cover slips. The second step of the procedure is to treat the cells with extraction buffer to remove cytoplasmic mRNA not bound to the er.

The next steps are to rapidly fix the cells and stain the mRNA by fluorescent in situ hybridization. The final steps are to image and then quantify the amount of fluorescence associated with the er. Ultimately, results can show how mRNAs are associated with the surface of this organelle under different conditions.

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