identify the transferred RNAs, as well as to measure the native
transcriptome of the acceptor cell. As a negative control, donor
and acceptor monocultures are mixed together after harvesting and
immediately sorted prior to RNA-seq analysis. A pre-validated
transferred mRNA is used as a positive control using reverse
transcription-polymerase chain reaction (RT-PCR) to first check
for the efficiency of cell sorting. This protocol is scalable to allow
for the rapid collection of large numbers of cells and can be applied
to study RNA transfer in systems having multiple cell types both
in vitro (e.g., organoids) and in vivo (e.g., tumor or tissue samples)
(see Notes 1 and 2).
2 Materials
The following protocol is established for the co-culture of 3 Â 10
6
human and mouse cells each per biological replicate (see Note 3). In
this protocol, the following specific cell types used are:
1. Mouse embryonic fibroblasts (MEFs) tagged with 24 repeats
of the MS2 coat protein (MCP)-binding sequence (MBS) RNA
aptamer between the ORF and the 3
0 UTR of both endogenous alleles of β-actin (referred to here as “MBS-MEFs”) (see
Fig. 1 and [21]): The β-actin-MBS gene serves as a specific
marker for the MEFs, while its message serves as a marker for
RNA transfer to the recipient human cells.
Fig. 1 Schematic of the MS2 aptamer-tagged β-actin alleles expressed in MBS-MEFs. A schematic representing aptamer tagging of both genomic loci of the mouse β-actin gene by insertion of MBS sequence in the
3
0 UTR [21]. The MBS sequence consists of 12 repeats of a unit, each unit consisting of a dimer of MS2 stemloops and three linker regions, thus containing 24 MS2 stem-loops in total. This MBS sequence is inserted
441 bp downstream of the stop codon of β-actin ORF. The expressed mRNA is shown as dashed arrow.
Fluorescent (Cy3)-labeled DNA probes complementary to the linker regions between each dimer of MS2 stemloops are used to detect β-actin-MBS mRNA by single-molecule FISH [21]. Forward (F) and reverse (R) primers
to detect the presence of the β-actin-MBS gene, as well as the smFISH probes are illustrated. TSS,
transcription start site; stop, stop codon; UTR, untranslated region; MBS, MS2 bacteriophage stem-loop
sequence/MS2 coat protein-binding sequence
Use of RNA Tagging as a Control for RNA Transferome Analysis
197
transcriptome of the acceptor cell. As a negative control, donor
and acceptor monocultures are mixed together after harvesting and
immediately sorted prior to RNA-seq analysis. A pre-validated
transferred mRNA is used as a positive control using reverse
transcription-polymerase chain reaction (RT-PCR) to first check
for the efficiency of cell sorting. This protocol is scalable to allow
for the rapid collection of large numbers of cells and can be applied
to study RNA transfer in systems having multiple cell types both
in vitro (e.g., organoids) and in vivo (e.g., tumor or tissue samples)
(see Notes 1 and 2).
2 Materials
The following protocol is established for the co-culture of 3 Â 10
6
human and mouse cells each per biological replicate (see Note 3). In
this protocol, the following specific cell types used are:
1. Mouse embryonic fibroblasts (MEFs) tagged with 24 repeats
of the MS2 coat protein (MCP)-binding sequence (MBS) RNA
aptamer between the ORF and the 3
0 UTR of both endogenous alleles of β-actin (referred to here as “MBS-MEFs”) (see
Fig. 1 and [21]): The β-actin-MBS gene serves as a specific
marker for the MEFs, while its message serves as a marker for
RNA transfer to the recipient human cells.
Fig. 1 Schematic of the MS2 aptamer-tagged β-actin alleles expressed in MBS-MEFs. A schematic representing aptamer tagging of both genomic loci of the mouse β-actin gene by insertion of MBS sequence in the
3
0 UTR [21]. The MBS sequence consists of 12 repeats of a unit, each unit consisting of a dimer of MS2 stemloops and three linker regions, thus containing 24 MS2 stem-loops in total. This MBS sequence is inserted
441 bp downstream of the stop codon of β-actin ORF. The expressed mRNA is shown as dashed arrow.
Fluorescent (Cy3)-labeled DNA probes complementary to the linker regions between each dimer of MS2 stemloops are used to detect β-actin-MBS mRNA by single-molecule FISH [21]. Forward (F) and reverse (R) primers
to detect the presence of the β-actin-MBS gene, as well as the smFISH probes are illustrated. TSS,
transcription start site; stop, stop codon; UTR, untranslated region; MBS, MS2 bacteriophage stem-loop
sequence/MS2 coat protein-binding sequence
Use of RNA Tagging as a Control for RNA Transferome Analysis
197