A Protocol for Non-biased Identification of RNAs Transferred Between Heterologous Mammalian Cell Types Using RNA Tagging, Cell Sorting, and Sequencing.
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.
['Dasgupta|Sandipan|S|', 'Gerst|Jeffrey E|JE|']
D000199:Actins D000818:Animals D001692:Biological Transport D002450:Cell Communication D002460:Cell Line D002469:Cell Separation D002478:Cells, Cultured D003001:Cloning, Molecular D018920:Coculture Techniques D059014:High-Throughput Nucleotide Sequencing D006801:Humans D017909:Levivirus D060526:Magnetic Fields D012333:RNA, Messenger D020133:Reverse Transcriptase Polymerase Chain Reaction D017423:Sequence Analysis, RNA
RNA transfer mRNA Membrane nanotubes Co-culture RNA sequencing MACS MS2 MS2-binding sequence Magnetic sorting lncRNA miRNA β-Actin