products are detected, the number of PCR cycles can be
increased. However, we do not recommend going higher
than 32 cycles. We also recommend to include a positive control for each primer pair (e.g., cDNA collected from
MBS-MEF or MCF7 single cultures).
12. This protocol is not suitable for analysis of RNA transfer using
single-cell RNA-seq. The detection of a low level of transferred
RNAs requires the isolation of a high amount of RNA from
multiple cells (Fig. 4) [13].
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).
References
1. Morel O, Toti F, Hugel B, Freyssinet JM
(2004) Cellular microparticles: a disseminated
storage pool of bioactive vascular effectors.
Curr Opin Hematol 11:156–164
2. Ramachandran S, Palanisamy V (2012) Horizontal transfer of RNAs: exosomes as mediators of intercellular communication. Wiley
Interdiscip Rev RNA 2:286–293
3. Kim G, LeBlanc ML, Wafula EK, dePamphilis
CW, Westwood JH (2014) Genomic-scale
exchange of mRNA between a parasitic plant
and its hosts. Science 345:808–881
4. Shimizu K, Shinga J, Yamasaki S,
Kawamura M, Do ¨rrie J et al (2015) Transfer
of mRNA encoding invariant NKT cell receptors imparts glycolipid specific responses to T
cells and γδT cells. PLoS One 10(6):e0131477
5. Chen J, Hu C, Pan P (2017) Extracellular
vesicle microRNA transfer in lung diseases.
Front Physiol 8:1028
6. Nguyen MA, Karunakaran D, Geoffrion M,
Cheng HS, Tandoc K et al (2018) Extracellular
vesicles secreted by atherogenic macrophages
transfer microRNA to inhibit cell migration.
Arterioscler Thromb Vasc Biol 38:49–63
7. Das S, Halushka MK (2015) Extracellular vesicle microRNA transfer in cardiovascular disease. Cardiovasc Pathol 24:199–206
8. Ma P, Pan Y, Li W, Sun C, Liu J, Xu T, Shu Y
(2017) Extracellular vesicles-mediated noncoding RNAs transfer in cancer. J Hematol
Oncol 10(1):57
9. Takahashi K, Yan IK, Wood J, Haga H, Patel T
(2014) Involvement of extracellular vesicle
long noncoding RNA (linc-VLDLR) in tumor
cell responses to chemotherapy. Mol Cancer
Res 10:1377–1387
10. Takahashi K, Yan IK, Kogure T, Haga H, Patel
T (2014) Extracellular vesicle-mediated transfer of long non-coding RNA ROR modulates
chemosensitivity in human hepatocellular cancer. FEBS Open Biol 4:458–467
11. Cai Q, Qiao L, Wang M, He B, Lin FM,
Palmquist J, Huang SD, Jin H (2018) Plants
send small RNAs in extracellular vesicles to
fungal pathogen to silence virulence genes. Science 360:1126–1129
12. Maida Y, Takakura M, Nishiuchi T,
Yoshimoto T, Kyo S (2015) Exosomal transfer
of functional small RNAs mediates cancerstroma communication in human endometrium. Cancer Med 5(2):304–314
13. Haimovich G, Ecker CM, Dunagin MC,
Eggan E, Raj A, Gerst JE, Singer RH (2017)
Intercellular mRNA trafficking via membrane
Use of RNA Tagging as a Control for RNA Transferome Analysis
213
increased. However, we do not recommend going higher
than 32 cycles. We also recommend to include a positive control for each primer pair (e.g., cDNA collected from
MBS-MEF or MCF7 single cultures).
12. This protocol is not suitable for analysis of RNA transfer using
single-cell RNA-seq. The detection of a low level of transferred
RNAs requires the isolation of a high amount of RNA from
multiple cells (Fig. 4) [13].
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).
References
1. Morel O, Toti F, Hugel B, Freyssinet JM
(2004) Cellular microparticles: a disseminated
storage pool of bioactive vascular effectors.
Curr Opin Hematol 11:156–164
2. Ramachandran S, Palanisamy V (2012) Horizontal transfer of RNAs: exosomes as mediators of intercellular communication. Wiley
Interdiscip Rev RNA 2:286–293
3. Kim G, LeBlanc ML, Wafula EK, dePamphilis
CW, Westwood JH (2014) Genomic-scale
exchange of mRNA between a parasitic plant
and its hosts. Science 345:808–881
4. Shimizu K, Shinga J, Yamasaki S,
Kawamura M, Do ¨rrie J et al (2015) Transfer
of mRNA encoding invariant NKT cell receptors imparts glycolipid specific responses to T
cells and γδT cells. PLoS One 10(6):e0131477
5. Chen J, Hu C, Pan P (2017) Extracellular
vesicle microRNA transfer in lung diseases.
Front Physiol 8:1028
6. Nguyen MA, Karunakaran D, Geoffrion M,
Cheng HS, Tandoc K et al (2018) Extracellular
vesicles secreted by atherogenic macrophages
transfer microRNA to inhibit cell migration.
Arterioscler Thromb Vasc Biol 38:49–63
7. Das S, Halushka MK (2015) Extracellular vesicle microRNA transfer in cardiovascular disease. Cardiovasc Pathol 24:199–206
8. Ma P, Pan Y, Li W, Sun C, Liu J, Xu T, Shu Y
(2017) Extracellular vesicles-mediated noncoding RNAs transfer in cancer. J Hematol
Oncol 10(1):57
9. Takahashi K, Yan IK, Wood J, Haga H, Patel T
(2014) Involvement of extracellular vesicle
long noncoding RNA (linc-VLDLR) in tumor
cell responses to chemotherapy. Mol Cancer
Res 10:1377–1387
10. Takahashi K, Yan IK, Kogure T, Haga H, Patel
T (2014) Extracellular vesicle-mediated transfer of long non-coding RNA ROR modulates
chemosensitivity in human hepatocellular cancer. FEBS Open Biol 4:458–467
11. Cai Q, Qiao L, Wang M, He B, Lin FM,
Palmquist J, Huang SD, Jin H (2018) Plants
send small RNAs in extracellular vesicles to
fungal pathogen to silence virulence genes. Science 360:1126–1129
12. Maida Y, Takakura M, Nishiuchi T,
Yoshimoto T, Kyo S (2015) Exosomal transfer
of functional small RNAs mediates cancerstroma communication in human endometrium. Cancer Med 5(2):304–314
13. Haimovich G, Ecker CM, Dunagin MC,
Eggan E, Raj A, Gerst JE, Singer RH (2017)
Intercellular mRNA trafficking via membrane
Use of RNA Tagging as a Control for RNA Transferome Analysis
213