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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230044	1/2	Homo sapiens	Blood plasma	SEC (non-commercial) UF	Herrero-Lorenzo, Marina	2023	75%
Study summary Full title Small RNAs in plasma extracellular vesicles define biomarkers of premanifest changes in Huntington’s disease All authors Marina Herrero-Lorenzo, Jesús Pérez-Pérez, Georgia Escaramís, Saül Martínez-Horta, Rocío Pérez-González, Elisa Rivas-Asensio, Jaime Kulisevsky, Ana Gámez-Valero, Eulàlia Martí Journal bioRxiv Abstract Despite the advances in the understanding of Huntington’s disease (HD), there is the need for mole (show more...)Despite the advances in the understanding of Huntington’s disease (HD), there is the need for molecular biomarkers to categorize mutation-carriers during the preclinical stage of the disease preceding the functional decline. Small RNAs (sRNAs) are a promising source of biomarkers since their expression levels are highly sensitive to pathobiological processes. Here, using an optimized method for plasma extracellular vesicles (EVs) purification and an exhaustive analysis pipeline of sRNA sequencing data, we show that EV-sRNAs are early downregulated in mutation-carriers, and that this deregulation is associated with premanifest cognitive performance. Seven candidate sRNAs (tRF-Glu-CTC, tRF-Gly-GCC, miR-451a, miR-21-5p, miR-26a-5p, miR-27a-3p, and let7a-5p) were validated in additional subjects, showing a significant diagnostic accuracy at premanifest stages. Of these, miR – 21-5p was significantly decreased over time in a longitudinal study; and miR-21-5p and miR-26a-5p levels correlated with cognitive changes in the premanifest cohort. In summary, the present results suggest that deregulated plasma EV-sRNAs define an early biosignature in mutation carriers with specific species sensing the progression and cognitive changes occurring at the premanifest stage. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Size-exclusion chromatography (non-commercial) Ultrafiltration Protein markers EV: Alix/ CD9/ Flotillin-1/ TSG101/ Syntenin/ CD63/ CD81 non-EV: Albumin/ COX4/ Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 20 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per mililiter of pooled fractions Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ Flotillin-1/ TSG101/ Syntenin Not detected contaminants Albumin/ COX4/ Calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNA-sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 146 EV concentration Yes Particle yield number of particles per 2mL of starting sample: 8.330E09 EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 111

	EV230044	2/2	Homo sapiens	Blood plasma	SEC (non-commercial) UF	Herrero-Lorenzo, Marina	2023	75%
Study summary Full title Small RNAs in plasma extracellular vesicles define biomarkers of premanifest changes in Huntington’s disease All authors Marina Herrero-Lorenzo, Jesús Pérez-Pérez, Georgia Escaramís, Saül Martínez-Horta, Rocío Pérez-González, Elisa Rivas-Asensio, Jaime Kulisevsky, Ana Gámez-Valero, Eulàlia Martí Journal bioRxiv Abstract Despite the advances in the understanding of Huntington’s disease (HD), there is the need for mole (show more...)Despite the advances in the understanding of Huntington’s disease (HD), there is the need for molecular biomarkers to categorize mutation-carriers during the preclinical stage of the disease preceding the functional decline. Small RNAs (sRNAs) are a promising source of biomarkers since their expression levels are highly sensitive to pathobiological processes. Here, using an optimized method for plasma extracellular vesicles (EVs) purification and an exhaustive analysis pipeline of sRNA sequencing data, we show that EV-sRNAs are early downregulated in mutation-carriers, and that this deregulation is associated with premanifest cognitive performance. Seven candidate sRNAs (tRF-Glu-CTC, tRF-Gly-GCC, miR-451a, miR-21-5p, miR-26a-5p, miR-27a-3p, and let7a-5p) were validated in additional subjects, showing a significant diagnostic accuracy at premanifest stages. Of these, miR – 21-5p was significantly decreased over time in a longitudinal study; and miR-21-5p and miR-26a-5p levels correlated with cognitive changes in the premanifest cohort. In summary, the present results suggest that deregulated plasma EV-sRNAs define an early biosignature in mutation carriers with specific species sensing the progression and cognitive changes occurring at the premanifest stage. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Huntington's disease Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Size-exclusion chromatography (non-commercial) Ultrafiltration Protein markers EV: Alix/ CD9/ Flotillin-1/ TSG101/ Syntenin/ CD63/ CD81 non-EV: Albumin/ COX4/ Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 20 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per mililiter of pooled fractions Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ Flotillin-1/ TSG101/ Syntenin Not detected contaminants Albumin/ COX4/ Calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNA-sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 80-300 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV230044
species	Homo sapiens
sample type	Blood plasma
condition	Control condition	Huntington's disease
separation protocol	Size-exclusion chromatography (non-commercial)/ Ultrafiltration	Size-exclusion chromatography (non-commercial)/ Ultrafiltration
Exp. nr.	1	2
EV-METRIC %	75	75