MiRNA references will continue to develop, but success will ultimately be depending on further standardization of EV isolation and information analysis. Based on the current data, we propose analysing reference transcripts in each and every study individually.JOURNAL OF EXTRACELLULAR VESICLESLBT02.Little extracellular vesicle content in porcine blood plasma, cerebrospinal fluid and seminal plasma for proteomic analyses in biomarker discovery Helena Kupcova Skalnikovaa, Jakub Cervenkaa, Karolina Turnovcovab, Bozena Bohuslavovaa, Jana Juhasovaa, Stefan Juhasa and Petr VodickaaaLBT02.Quantitative proteomic profiling of tissue-exudative EVs identified a novel diagnostic antigen for early detection of colorectal cancer Makoto Konishia, Makoto Sumazakia, Satoshi Nagayamab and Koji Uedaa Cancer Proteomics Group, Cancer Precision Medicine Center, Japanese Foundation for Cancer Investigation, Tokyo, Japan; bDepartment of Gastroenterological Surgery, Cancer Fc epsilon RII/CD23 Proteins web Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, JapanaCzech Academy of Sciences, Institute of Animal Physiology and TFR-1/CD71 Proteins MedChemExpress Genetics, Libechov, Czech Republic, Libechov, Czech Republic; bCzech Academy of Sciences, Institute of Experimental Medicine, Prague, Czech Republic, Prague, Czech RepublicIntroduction: Extracellular vesicles (EVs) released to body fluids carry molecules on the source cells and are subjects of intensive research of protein and nucleic acid biomarkers of ailments. Pig represents a important experimental biomedical model to study human ailments due to close anatomic and physiologic similarity to human. The aim of this perform was to examine suitability of porcine blood plasma, cerebrospinal fluid and seminal plasma for EV isolation for proteomic analyses and optimize sample preparation for mass spectrometry. Solutions: EVs were isolated from porcine physique fluids by differential centrifugation and ultracentrifugation and characterized by transmission electron microscopy, flow cytometry and western blotting. 3 different lysis buffers (RIPA, Triton X100 and SDS) have been compared in efficacy to extract EV proteins in mixture with filter-aided sample preparation (FASP) for LCMS/MS evaluation (triple TOF). Outcomes: Seminal plasma yielded biggest quantity of EVs, followed by blood plasma. In cerebrospinal fluid, the EV content was very low. Proteomic evaluation of seminal plasma-derived EVs enabled identification of approximately 1200 proteins, including 76 of your top 100 mainly identified proteins in EVs (Exocarta). About 550 proteins have been quantified by SWATH-MS. In contrast, only 200 proteins have been identified in the crude seminal plasma employed for EV isolation. Summary/conclusion: We’ve got optimized methods for the EV enrichment from porcine physique fluids and for characterization of their protein content by mass spectrometry. Such tactics may be applied to biomarker discovery in porcine model of diseases as well as adopted to other species, like human. Funding: This study was supported by Czech Science Foundation (reg. No. 19-01747S), Operational Programme Investigation, Development and Education (reg. No. CZ.02.1.01/0.0/0.0/16_019/0000785), and National Sustainability Programme I. of the Czech Ministry of Education, Youth and Sports (reg. No. LO1609).Introduction: Improvement of biomarkers for early detection of colorectal cancer (CRC) is demanded as the number of CRC individuals is escalating. Current studies exhibit that extracellular vesicles (EVs) are expected as biomarker carriers in any.