2a66fe7d3be7c6aa5b41990f201063e8 320155.pdf 7118ce62a8b4eb9dca9284c612b39df17a2f8e28 320155.pdf f901f7116682e0040426423a5706833f494d026988b9952145357541cb032756 320155.pdf Title: Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing Subject: MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples. Keywords: extracellular vesicle isolation; miRNA sequencing; diagnostic biomarker Author: Meritxell Llorens-Revull, Brenda Martínez-González, Josep Quer, Juan Ignacio Esteban, Gonzalo Núñez-Moreno, Pablo Mínguez, Idoia Burgui, Ricardo Ramos-Ruíz, María Eugenia Soria, Angie Rico, Mar Riveiro-Barciela, Silvia Sauleda, María Piron, Irene Corrales, Francesc E. Borràs, Francisco Rodríguez-Frías, Ariadna Rando, Clara Ramírez-Serra, Silvia Camós, Esteban Domingo, Marta Bes, Celia Perales and Maria Isabel Costafreda Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Aug 1 09:27:58 2023 CEST ModDate: Tue Aug 1 09:32:25 2023 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1923528 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OPNYWB+VnURWPalladioL Type 1 Custom yes yes no 10 0 YCZOHB+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 DULVXQ+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 NABHTA+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 CCDETI+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 46 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 49 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 71 0 CCOJMN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 85 0 CCOJMO+PalatinoLinotype CID TrueType Identity-H yes yes yes 88 0 CCOJMP+PalatinoLinotype TrueType WinAnsi yes yes no 94 0 CCOJNB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 97 0 CCOKPL+PalatinoLinotype TrueType MacRoman yes yes no 100 0 IFEDSM+CMSY10 Type 1 Builtin yes yes no 107 0 CCOJMN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 121 0 CCOJMO+PalatinoLinotype CID TrueType Identity-H yes yes yes 124 0 CCOJMP+PalatinoLinotype TrueType WinAnsi yes yes no 130 0 CCOJNB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 133 0 CCOKPL+PalatinoLinotype TrueType MacRoman yes yes no 136 0 CCOJMN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 154 0 CCOJMO+PalatinoLinotype CID TrueType Identity-H yes yes yes 157 0 CCOJMP+PalatinoLinotype TrueType WinAnsi yes yes no 163 0 CCOJNB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 166 0 CCOKPL+PalatinoLinotype TrueType MacRoman yes yes no 169 0 CCOJMN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 184 0 CCOJMO+PalatinoLinotype CID TrueType Identity-H yes yes yes 187 0 CCOJMP+PalatinoLinotype TrueType WinAnsi yes yes no 193 0 CCOJNB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 196 0 CCOKPL+PalatinoLinotype TrueType MacRoman yes yes no 199 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-10-08 03:54:41 CEST RepresentationInformation: 320155.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-10-07 08:44:25 CEST Size: 1923528 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 400 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: Comparative Characterization of Three EV Isolation Methods Destination: subsection.2.1 Item: Title: miRNA Library Evaluation for Next-Generation Sequencing Destination: subsection.2.2 Item: Title: Selection of an EV Isolation Method According to miRNA Sequence Recovery Destination: subsection.2.3 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Plasma Isolation Destination: subsection.4.1 Item: Title: Cell Debris, Apoptotic Bodies, and Microvesicle Removal Destination: subsection.4.2 Item: Title: Extracellular Vesicle Isolation Methods Destination: subsection.4.3 Item: Title: EV Characterization Destination: subsection.4.4 Item: Title: RNA Extraction Destination: subsection.4.5 Item: Title: Library Preparation, Size Selection, and Sequencing Destination: subsection.4.6 Item: Title: Bioinformatics Analyses Destination: subsection.4.7 Item: Title: Statistics Destination: subsection.4.8 Item: Title: References Destination: section.A. Info: Title: Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing Author: Meritxell Llorens-Revull, Brenda Martínez-González, Josep Quer, Juan Ignacio Esteban, Gonzalo Núñez-Moreno, Pablo Mínguez, Idoia Burgui, Ricardo Ramos-Ruíz, María Eugenia Soria, Angie Rico, Mar Riveiro-Barciela, Silvia Sauleda, María Piron, Irene Corrales, Francesc E. Borràs, Francisco Rodríguez-Frías, Ariadna Rando, Clara Ramírez-Serra, Silvia Camós, Esteban Domingo, Marta Bes, Celia Perales and Maria Isabel Costafreda Subject: MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples. Keywords: extracellular vesicle isolation; miRNA sequencing; diagnostic biomarker Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Aug 01 09:27:58 CEST 2023 ModDate: Tue Aug 01 09:32:25 CEST 2023 ID: 0xdd86aa9a89f6d57a4f94711c7973ebe3, 0xdd86aa9a89f6d57a4f94711c7973ebe3 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: DULVXQ+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 250 FontDescriptor: FontName: DULVXQ+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: NABHTA+URWPalladioL-Ital FontSubset: true FirstChar: 43 LastChar: 122 FontDescriptor: FontName: NABHTA+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true 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