A novel precipitation-based protocol of extracellular vesicles isolation from biological fluids for routine diagnosis of prostate cancer

Maria Konoshenko ^1,2 Evgeniy Lekchnov ^1,2 Olga Bryzgunova ^1,2 Elena Kiseleva ⁵ Ivan Zaporozhchenko ^1,2 Elena Rykova ^1,2 Sergey Yarmoschuk ³ Oxana Pashkovskaya ³ Anton Gorizkii ³ Svetlana Pak ⁴ Pavel Laktionov ^1,2

¹The Laboratory of Molecular Medicine, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia
²The Laboratory of Biomedical Technologies, Meshalkin Siberian Federal Biomedical Research Center, Ministry of Public Health of the Russian Federation, Novosibirsk, Russia
³Department of Oncology and Radiotherapy, Meshalkin Siberian Federal Biomedical Research Center, Ministry of Public Health of the Russian Federation, Novosibirsk, Russia
⁴The Department of Blood Transfusion, Meshalkin Siberian Federal Biomedical Research Center, Ministry of Public Health of the Russian Federation, Novosibirsk, Russia
⁵Cell Structural Biology Sector, Federal Research Center, Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia

The research towards the discovery of extracellular vesicles (EV)-based biomarkers has induced a great interest and a vast number of researches in biology and medicine. Still, there is no consensus according to what protocol should be used to isolate EVs for scientific and clinical use. The aim of this work was to develop a simple and effective method for EVs isolation suitable for scientific and clinical analysis of miRNAs packed in EVs. The samples of urine and blood of the healthy donors and prostate cancer patients were used. Ultracentrifugation was used as a gold standard of EVs isolation.

A sample of biological fluid was first subjected to low-speed centrifugation. To reduce the number of supramolecular aggregates and to exclude their coprecipitation the supernatants were diluted with NaCl and Tris HCl pH7.0. To induce the aggregation of EVs Dextran Blue was added and EVs were precipitated by addition of low concentrated polyethylene glycol and low-speed centrifugation. The method provides precipitation of membrane coated EVs mainly, rather than membrane-free microparticles, large biopolymers, and supramolecular complexes. The EVs fractions were characterized by TEM; miRNAs were isolated and quantified by RT-TaqMan-PCR. Both isolates contain EVs but mainly their aggregates when precipitation protocol was used. Ultracentrifugation and precipitation protocols resulted in equal detection of mir-19b, mir-378a, miR-425 in urine and miR-378a in plasma EVs, whereas precipitation-based EV isolation from plasma yielded significantly higher miR-19b, miR-425 than ultracentrifugation.

The precipitation method is inexpensive, fast and simple, doesn’t need complicated equipment, can be adapted for EVs isolation from different samples. The method enables to process a large number of samples simultaneously and to isolate EVs free from RT-PCR inhibitors. Thus our precipitation method represents the convenient protocol of EVs isolation for both research and clinical laboratories.

The work was supported by the Russian Science Foundation (no. 16-15-00124).