|
Autor/a:
|
Campoy, Irene; Lanau, Lucia; Altadill, Tatiana; Sequeiros, Tamara; Cabrera, Silvia; Cubo Abert, Montserrat; Pérez Benavente, Assumpción; Garcia, Angel; Borrós i Gómez, Salvador; Santamaria, Anna; Ponce, Jordi; Matias-Guiu, Xavier; Reventós, Jaume; Gil-Moreno, Antonio; Rigau, Marina; Colás, Eva
|
|
Notas:
|
Background: Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications
could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become
an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study
was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols
for their isolation, characterization, and analysis.
Methods: We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases.
The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential
centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol.
Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot.
Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of
the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples.
Results: All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best
performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed
a higher concentration of vesicles with a mode of 135 ± 5 nm, and immunoblot showed a higher expression of
exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained
in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion
with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR.
Conclusion: We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were
successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further
analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not
ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future
comparative studies to foster the field of biomarker research in gynecology.
Keywords: Biomarker, Endometrial biopsy, Exosomes, Exosomes isolation protocols, Exosome-like vesicles,
Extracellular vesicles, Gynecological disorders, Microvesicles, RNA, Uterine aspirates
This work was supported by the Spanish Ministry of Health (RD12/0036/0035), the Spanish Ministry of Economy and Competitivity (PI14/02043), the “Fondo Europeo de Desarrollo Regional” FEDER (RTC-2014-3110-1), the AECC (Grupos Estables de Investigacion 2011 - AECC- GCB 110333 REVE), the Fundació La Marató TV3 (2/C/2013), the CIRIT Generalitat de Catalunya (2014 SGR 1330) and the European Commission, 7th Framework Programe, IRSES (PROTBIOFLUID –269285)—Belgium. AGAUR (2014F1_B1 00014) granted Ms. Lanau, the Instituto Carlos III (Spanish Ministry of Economy and Competitivity) supported Dr. Santamaria (CP13/00158) by the Miguel Servet program, and the Spanish Ministry of Economy and Competitiveness (FPDI-2013-18322) granted Dr. Colás. |
