Visualization of Endoplasmic Reticulum-Associated mRNA in Mammalian Cells

In eukaryotes, most mRNAs that encode secretory or membrane-bound proteins are translated by ribosomes associated with the surface of the endoplasmic reticulum (ER). Other such mRNAs are tethered to the ER by mRNA receptors. However, there has been much debate as to whether all mRNAs, regardless of their encoded polypeptide, are anchored to the ER at some low level. Here we describe a protocol to visualize ER-associated mRNAs in tissue culture cells by single-molecule fluorescence in situ hybridization (smFISH). Using this protocol, we have established that a subset of all mRNAs, regardless of whether they encode secretory or cytosolic proteins, are ER associated in a ribosome-dependent manner.