Detection of mRNA Transfer Between Mammalian Cells in Coculture by Single-Molecule Fluorescent In Situ Hybridization (smFISH)
In eukaryotic cells, a small percentage of mRNA molecules can undergo transfer from one cell to another. mRNA transfer occurs primarily via membrane nanotubes, which are long thin protrusions that are produced by numerous cell types and can connect cells that can be up to hundreds of microns apart. Potentially, mRNAs might also transfer via extracellular vesicles (EVs). Here we describe a method to detect transferred mRNA in cocultures of two different cell types and to distinguish between nanotube- and EVs-mediated transfer. This method uses single molecule fluorescent in situ hybridization (smFISH) to provide an accurate and quantitative detection of transferred mRNA molecules and their subcellular localization. Following the guidelines presented here will allow the user to investigate mRNA transfer of most transcripts in any co-culture system. In addition, we present modifications that improve nanotube preservation during the smFISH procedure.
Key wordsmRNA Membrane nanotubes Extracellular vesicles Exosomes Single-molecule fluorescent in situ hybridization β-Actin Glutaraldehyde
G.H. was funded by the Koshland Foundation and McDonald-Leapman Grant Senior Postdoctoral Fellowships. This work was funded by grants to J.E.G. from the Joel and Mady Dukler Fund for Cancer Research, the Jean-Jacques Brunschwig Fund for the Molecular Genetics of Cancer, a Proof-of-Principle Grant from the Moross Integrated Cancer Center (Weizmann Institute of Science), and the US-Israel Binational Science Foundation-National Science Foundation (#2015846).
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