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RNA Tagging pp 195–214Cite as

A Protocol for Non-biased Identification of RNAs Transferred Between Heterologous Mammalian Cell Types Using RNA Tagging, Cell Sorting, and Sequencing

Part of the Methods in Molecular Biology book series (MIMB,volume 2166)

Abstract

Intercellular communication is a major hallmark of multicellular organisms and is responsible for coordinating cell and tissue differentiation, immune responses, synaptic transmission, and both paracrine and endocrine signaling, for example. Small molecules, peptides, and proteins have all been studied extensively as mediators of intercellular communication; however, RNAs have also been shown recently to transfer between cells. In mammalian cells, microRNAs, tRNAs, short noncoding RNAs, mRNA fragments, as well as full-length mRNAs have all been shown to transfer between cells either by exosomes or by membrane nanotubes. We have previously described nanotube-mediated cell-cell transfer of specific mRNAs between heterologous mammalian cell types cultured in vitro. Here, we describe a simple method for the unbiased and quantitative identification of the complete range of transferred mRNAs (i.e., the mRNA transferome) in one population of mammalian cells following co-culture with another population. After co-culture, the individual cell populations are sorted by magnetic bead-mediated cell sorting and the transferred RNAs are then identified by downstream analysis methods, such as RNA sequencing. Application of this technique not only allows for determination of the mRNA transferome, but can also reveal changes in the native transcriptome of a cell population after co-culture. This can indicate the effect that co-culture and intercellular transfer of mRNA have upon cell physiology.

Key words

  • mRNA
  • MS2
  • MS2-binding sequence
  • β-Actin
  • Magnetic sorting
  • MACS
  • RNA sequencing
  • Co-culture
  • RNA transfer
  • Membrane nanotubes
  • miRNA
  • lncRNA

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Acknowledgments

The authors thank Tsviya Olender for the simulation of human and mouse “read” alignments. 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), the German-Israel Foundation (GIF; I-1461-412.13/2018) and the US-Israel Binational Science Foundation-National Science Foundation (#2015846).

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Correspondence to Jeffrey E. Gerst .

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Dasgupta, S., Gerst, J.E. (2020). A Protocol for Non-biased Identification of RNAs Transferred Between Heterologous Mammalian Cell Types Using RNA Tagging, Cell Sorting, and Sequencing. In: Heinlein, M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0712-1_11

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  • DOI: https://doi.org/10.1007/978-1-0716-0712-1_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0711-4

  • Online ISBN: 978-1-0716-0712-1

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