1af63b26957fae7f45de791289756764 ijms_25_13705_v2.pdf 829f828ca3351f7a4ad3155521184c2b505033d7 ijms_25_13705_v2.pdf b52d699e5abebfacb347b71969f311ca90885568f160ea18ce2897034d9cd38e ijms_25_13705_v2.pdf Title: Methodological Assessment of ExoGAG for Isolation of Cerebrospinal Fluid Extracellular Vesicles as a Source of Biomarkers Subject: Extracellular vesicles (EVs) in cerebrospinal fluid (CSF) represent a valuable source of biomarkers for central nervous system (CNS) diseases, offering new pathways for diagnosis and monitoring. However, existing methods for isolating EVs from CSF often prove to be labor-intensive and reliant on specialized equipment, hindering their clinical application. In this study, we present a novel, clinically compatible method for isolating EVs from CSF. We optimized the use of ExoGAG, a commercially available reagent that has been tested in plasma, urine and semen, and compared it directly with differential ultracentrifugation using Western blotting, protein quantification, nanoparticle tracking analysis, and cryogenic electron microscopy. Additionally, we analyzed the presence of specific microRNAs (miRNAs) known to be present in CSF-derived EVs. Our data demonstrate that ExoGAG is an effective method for isolating EVs from CSF, yielding a higher amount of EVs compared to traditional ultracentrifugation methods, and with comparable levels of specific miRNAs. In conclusion, the use of ExoGAG in a clinical setting may facilitate the testing of biomarkers essential for tracking brain pathology in CNS diseases. Keywords: extracellular vesicles; cerebrospinal fluid; isolation method; ultracentrifugation; ExoGAG; biomarkers; miRNAs Author: Nil Salvat-Rovira, Anna Vazquez-Oliver, Elisa Rivas-Asensio, Marina Herrero-Lorenzo, Ana Gámez-Valero, Jesús Pérez-Pérez, Cristina Izquierdo, Antonia Campolongo, Eulàlia Martí, Jaime Kulisevsky and Rocío Pérez-González Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Tue Dec 24 04:57:45 2024 CET ModDate: Tue Jan 14 14:28:15 2025 CET Custom Metadata: no Metadata Stream: yes Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1844620 bytes Optimized: yes PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- DDRAXE+VnURWPalladioL-Bold Type 1 Custom yes yes yes 233 0 MGZXPN+URWPalladioL-Bold Type 1 Custom yes yes yes 237 0 WKGVDR+URWPalladioL-Roma 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Identity-H yes yes yes 116 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 124 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 127 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 130 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 136 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-01-15 02:41:47 CET RepresentationInformation: ijms_25_13705_v2.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-01-14 14:33:08 CET Size: 1844620 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf Profile: Linearized PDF PDFMetadata: Objects: 0 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Evaluation of ExoGAG for CSF EV Isolation Destination: subsection.2.1 Item: Title: Head-to-Head Comparison of ExoGAG and Ultracentrifugation (UC) Methods Destination: subsection.2.2 Item: Title: Evaluation of miRNA Cargo in EVs Destination: subsection.2.3 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Collection of CSF and EV Isolation Destination: subsection.4.1 Item: Title: Sample Homogenization, Protein Estimation, and Western Blot Analyses Destination: subsection.4.2 Item: Title: Nanoparticle Tracking Analyses (NTA) Destination: subsection.4.3 Item: Title: Cryogenic Electron Microscopy (Cryo-EM) Destination: subsection.4.4 Item: Title: RNA Extraction Destination: subsection.4.5 Item: Title: qPCR of miRNAs Destination: subsection.4.6 Item: Title: Statistical Analysis Destination: subsection.4.7 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.6 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: WKGVDR+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 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DDRAXE+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: MGZXPN+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 135 FontDescriptor: FontName: MGZXPN+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: YFFKLH+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: YFFKLH+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true XMP: application/pdf Nil Salvat-Rovira, Anna Vazquez-Oliver, Elisa Rivas-Asensio, Marina Herrero-Lorenzo, Ana Gámez-Valero, Jesús Pérez-Pérez, Cristina Izquierdo, Antonia Campolongo, Eulàlia Martí, Jaime Kulisevsky and Rocío Pérez-González Extracellular vesicles (EVs) in cerebrospinal fluid (CSF) represent a valuable source of biomarkers for central nervous system (CNS) diseases, offering new pathways for diagnosis and monitoring. However, existing methods for isolating EVs from CSF often prove to be labor-intensive and reliant on specialized equipment, hindering their clinical application. In this study, we present a novel, clinically compatible method for isolating EVs from CSF. We optimized the use of ExoGAG, a commercially available reagent that has been tested in plasma, urine and semen, and compared it directly with differential ultracentrifugation using Western blotting, protein quantification, nanoparticle tracking analysis, and cryogenic electron microscopy. Additionally, we analyzed the presence of specific microRNAs (miRNAs) known to be present in CSF-derived EVs. Our data demonstrate that ExoGAG is an effective method for isolating EVs from CSF, yielding a higher amount of EVs compared to traditional ultracentrifugation methods, and with comparable levels of specific miRNAs. In conclusion, the use of ExoGAG in a clinical setting may facilitate the testing of biomarkers essential for tracking brain pathology in CNS diseases. 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