KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine

Tutkimustuotos: ArtikkelijulkaisuArtikkeliTieteellinenvertaisarvioitu

Kuvaus

Urinary extracellular vesicles (EVs) are a promising source of biomarkers, which can be obtained in a non-invasive manner. However, the yield of EVs from urine samples may be insufficient for various analyses due to the entrapment of EVs by the Tamm-Horsfall protein (THP) meshwork. Here, we developed a simple dilution protocol to increase the urinary EV yield by disrupting the interaction between THP filaments and EVs with the help of alkaline pH and lowered ionic concentration. The integrity of the EVs and THP was assessed by electron microscopy. The effect of the protocol on the EV yield was quantified against an undiluted control by western blotting of four EV markers, nanoparticle tracking analysis and measuring of the RNA/miRNA concentration of the EV samples. The average EV yield from the dilution protocol was 2-7 fold the yield from the undiluted control i.e. increased by 130-624% as measured by western blotting and NTA. The yield increased most from samples with a high THP to EV ratio. The morphology and size range of the EVs were unaltered by the protocol. However, RNA/miRNA yields were the same as from the undiluted control and THP filaments could still be detected in EV samples. The dilution protocol, that we named KeepEX, provides a simple and efficient way to prevent loss of EVs thus increasing their yield from urine. Since KeepEX does not require individual adjustment of sample pH nor extra centrifugation steps, it could be used on its own or in combination with other EV purification protocols to improve EV isolation particularly from small urine volumes.
Alkuperäiskielienglanti
LehtiEuropean Journal of Pharmaceutical Sciences
Vuosikerta98
Sivut30-39
Sivumäärä10
ISSN0928-0987
DOI - pysyväislinkit
TilaJulkaistu - 15 helmikuuta 2017
OKM-julkaisutyyppiA1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä, vertaisarvioitu

Lisätietoja

LR: 20161125; CI: Copyright A(c) 2016; JID: 9317982; OTO: NOTNLM; 2016/05/30 [received]; 2016/10/11 [revised]; 2016/10/18 [accepted]; aheadofprint
Volume:
Proceeding volume:

Tieteenalat

  • 1182 Biokemia, solu- ja molekyylibiologia

Lainaa tätä

@article{812c1253a7b3445db35883a78a8c0d42,
title = "KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine",
abstract = "Urinary extracellular vesicles (EVs) are a promising source of biomarkers, which can be obtained in a non-invasive manner. However, the yield of EVs from urine samples may be insufficient for various analyses due to the entrapment of EVs by the Tamm-Horsfall protein (THP) meshwork. Here, we developed a simple dilution protocol to increase the urinary EV yield by disrupting the interaction between THP filaments and EVs with the help of alkaline pH and lowered ionic concentration. The integrity of the EVs and THP was assessed by electron microscopy. The effect of the protocol on the EV yield was quantified against an undiluted control by western blotting of four EV markers, nanoparticle tracking analysis and measuring of the RNA/miRNA concentration of the EV samples. The average EV yield from the dilution protocol was 2-7 fold the yield from the undiluted control i.e. increased by 130-624{\%} as measured by western blotting and NTA. The yield increased most from samples with a high THP to EV ratio. The morphology and size range of the EVs were unaltered by the protocol. However, RNA/miRNA yields were the same as from the undiluted control and THP filaments could still be detected in EV samples. The dilution protocol, that we named KeepEX, provides a simple and efficient way to prevent loss of EVs thus increasing their yield from urine. Since KeepEX does not require individual adjustment of sample pH nor extra centrifugation steps, it could be used on its own or in combination with other EV purification protocols to improve EV isolation particularly from small urine volumes.",
keywords = "Exosomes, Extracellular vesicles, THP, Urine, Yield, 1182 Biochemistry, cell and molecular biology",
author = "M. Puhka and Nordberg, {M. E.} and S. Valkonen and A. Rannikko and O. Kallioniemi and P. Siljander and H{\"a}llstr{\"o}m, {T. M. Af}",
note = "LR: 20161125; CI: Copyright A(c) 2016; JID: 9317982; OTO: NOTNLM; 2016/05/30 [received]; 2016/10/11 [revised]; 2016/10/18 [accepted]; aheadofprint Volume: Proceeding volume:",
year = "2017",
month = "2",
day = "15",
doi = "10.1016/j.ejps.2016.10.021",
language = "English",
volume = "98",
pages = "30--39",
journal = "European Journal of Pharmaceutical Sciences",
issn = "0928-0987",
publisher = "Elsevier Scientific Publ. Co",

}

KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine. / Puhka, M.; Nordberg, M. E.; Valkonen, S.; Rannikko, A.; Kallioniemi, O.; Siljander, P.; Hällström, T. M. Af.

julkaisussa: European Journal of Pharmaceutical Sciences, Vuosikerta 98, 15.02.2017, s. 30-39.

Tutkimustuotos: ArtikkelijulkaisuArtikkeliTieteellinenvertaisarvioitu

TY - JOUR

T1 - KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine

AU - Puhka, M.

AU - Nordberg, M. E.

AU - Valkonen, S.

AU - Rannikko, A.

AU - Kallioniemi, O.

AU - Siljander, P.

AU - Hällström, T. M. Af

N1 - LR: 20161125; CI: Copyright A(c) 2016; JID: 9317982; OTO: NOTNLM; 2016/05/30 [received]; 2016/10/11 [revised]; 2016/10/18 [accepted]; aheadofprint Volume: Proceeding volume:

PY - 2017/2/15

Y1 - 2017/2/15

N2 - Urinary extracellular vesicles (EVs) are a promising source of biomarkers, which can be obtained in a non-invasive manner. However, the yield of EVs from urine samples may be insufficient for various analyses due to the entrapment of EVs by the Tamm-Horsfall protein (THP) meshwork. Here, we developed a simple dilution protocol to increase the urinary EV yield by disrupting the interaction between THP filaments and EVs with the help of alkaline pH and lowered ionic concentration. The integrity of the EVs and THP was assessed by electron microscopy. The effect of the protocol on the EV yield was quantified against an undiluted control by western blotting of four EV markers, nanoparticle tracking analysis and measuring of the RNA/miRNA concentration of the EV samples. The average EV yield from the dilution protocol was 2-7 fold the yield from the undiluted control i.e. increased by 130-624% as measured by western blotting and NTA. The yield increased most from samples with a high THP to EV ratio. The morphology and size range of the EVs were unaltered by the protocol. However, RNA/miRNA yields were the same as from the undiluted control and THP filaments could still be detected in EV samples. The dilution protocol, that we named KeepEX, provides a simple and efficient way to prevent loss of EVs thus increasing their yield from urine. Since KeepEX does not require individual adjustment of sample pH nor extra centrifugation steps, it could be used on its own or in combination with other EV purification protocols to improve EV isolation particularly from small urine volumes.

AB - Urinary extracellular vesicles (EVs) are a promising source of biomarkers, which can be obtained in a non-invasive manner. However, the yield of EVs from urine samples may be insufficient for various analyses due to the entrapment of EVs by the Tamm-Horsfall protein (THP) meshwork. Here, we developed a simple dilution protocol to increase the urinary EV yield by disrupting the interaction between THP filaments and EVs with the help of alkaline pH and lowered ionic concentration. The integrity of the EVs and THP was assessed by electron microscopy. The effect of the protocol on the EV yield was quantified against an undiluted control by western blotting of four EV markers, nanoparticle tracking analysis and measuring of the RNA/miRNA concentration of the EV samples. The average EV yield from the dilution protocol was 2-7 fold the yield from the undiluted control i.e. increased by 130-624% as measured by western blotting and NTA. The yield increased most from samples with a high THP to EV ratio. The morphology and size range of the EVs were unaltered by the protocol. However, RNA/miRNA yields were the same as from the undiluted control and THP filaments could still be detected in EV samples. The dilution protocol, that we named KeepEX, provides a simple and efficient way to prevent loss of EVs thus increasing their yield from urine. Since KeepEX does not require individual adjustment of sample pH nor extra centrifugation steps, it could be used on its own or in combination with other EV purification protocols to improve EV isolation particularly from small urine volumes.

KW - Exosomes

KW - Extracellular vesicles

KW - THP

KW - Urine

KW - Yield

KW - 1182 Biochemistry, cell and molecular biology

U2 - 10.1016/j.ejps.2016.10.021

DO - 10.1016/j.ejps.2016.10.021

M3 - Article

VL - 98

SP - 30

EP - 39

JO - European Journal of Pharmaceutical Sciences

JF - European Journal of Pharmaceutical Sciences

SN - 0928-0987

ER -