الفهرس 
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Abstract
Cell free nucleic acids can be isolated from the serum of patients with various diseases and can be used as potential biomarkers. However nucleic acids are not stable enough for processing and many isolation protocols result in poor yields. Exosomes are small lipid bilayer vesicles that are defined by their endocytic origin and size range of 30–140 nm. They are constantly produced by different cell types and by both healthy and abnormal cells, and can be isolated from almost all body fluids. These vesicles, loaded with unique DNA, RNA and protein cargo, have a wide range of biological functions. characterization of the content profile of exosomes may reflect the state of the cells that release them, and this could be predictive of disease. However, Isolation of extracellular vesicles from plasma remains challenging due to the presence of contaminating plasma proteins and lipoproteins. This challenge hinders their implementation into translational tools due to the incomplete characterization of the protein composition of plasma-derived vesicles, in the size range of exosomes, as mass spectrometric analysis of plasma sub-components is noticeably troublesome. We have addressed this challenge by analyses of plasma-derived vesicles obtained by 2 coupled methodologies: Ultracentrifugation (UC) coupled with size-exclusion chromatography (SEC) to isolate and subsequently enrich exosomes. Our results support the use of UC coupled with SEC to obtain preparations of extracellular vesicles, from plasma that provide the best yield of proteins enriched in exosomes by liquid chromatography mass spectrometry (LC-MS) analyses with only a minimal amount of contaminants. The aim of this study was to extract and analyse nucleic acids from Exosomes in the plasma of human patients to be used as diagnostic biomarkers. In order to achieve this we had to develop a robust method to isolate and characterize Exosomes from plasma with minimal contamination by plasma proteins and lipoprotein particles. In this study, we isolated plasma exosomes from 15 patients with melanoma and 15 healthy individuals using the coupled ultracentrifugation and SEC method to explore the potential biomarkers for malignant melanoma. Two miRNAs; miRNA-221 and miRNA-149-3p, were subsequently detected in each sample by quantitative real time-PCR. Overall, these results indicate that plasma exosomal miRNAs, especially exo-miRNA-149-3p and exo-miRNA-221, have the potential to be used for diagnosis of melanoma in a clinical settingThis study demonstrates that a two-step isolation methodology, combining UC followed by SEC, isolates exosomes, from human plasma, and efficiently separates exosomes from the main contaminating plasma proteins and lipoproteins, as evidenced by characterization of protein content by proteomic characterization by MS. We subsequently isolated nucleic acids from exosomes and concluded that miRNA-149-3p and miRNA-221 were over-expressed in malignant melanoma patients compared to normal controls and can be used as potential biomarkers for malignant melanoma. After managing to establish a proper and stable methodology for isolating exosomes from human plasma, we recommend that this technique is used for isolating miRNAs from other tumors to be used as biomarkers for their early diagnosis.