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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220405	1/3	Homo sapiens	Cerebrospinal Fluid	UF qEV	Ursula S Sandau	2022	75%
Study summary Full title Differential Effects of APOE Genotype on MicroRNA Cargo of Cerebrospinal Fluid Extracellular Vesicles in Females With Alzheimer's Disease Compared to Males All authors Ursula S Sandau, Trevor J McFarland, Sierra J Smith, Douglas R Galasko, Joseph F Quinn, Julie A Saugstad Journal Abstract Multiple biological factors, including age, sex, and genetics, influence Alzheimer's disease (AD) ri (show more...)Multiple biological factors, including age, sex, and genetics, influence Alzheimer's disease (AD) risk. Of the 6.2 million Americans living with Alzheimer's dementia in 2021, 3.8 million are women and 2.4 million are men. The strongest genetic risk factor for sporadic AD is apolipoprotein E-e4 (APOE-e4). Female APOE-e4 carriers develop AD more frequently than age-matched males and have more brain atrophy and memory loss. Consequently, biomarkers that are sensitive to biological risk factors may improve AD diagnostics and may provide insight into underlying mechanistic changes that could drive disease progression. Here, we have assessed the effects of sex and APOE-e4 on the miRNA cargo of cerebrospinal fluid (CSF) extracellular vesicles (EVs) in AD. We used ultrafiltration (UF) combined with size exclusion chromatography (SEC) to enrich CSF EVs (e.g., Flotillin+). CSF EVs were isolated from female and male AD or controls (CTLs) that were either APOE-e3,4 or -e3,3 positive (n = 7/group, 56 total). MiRNA expression levels were quantified using a custom TaqMan™ array that assayed 190 miRNAs previously found in CSF, including 25 miRNAs that we previously validated as candidate AD biomarkers. We identified changes in the EV miRNA cargo that were affected by both AD and sex. In total, four miRNAs (miR-16-5p, -331-3p, -409-3p, and -454-3p) were significantly increased in AD vs. CTL, independent of sex and APOE-e4 status. Pathway analysis of the predicted gene targets of these four miRNAs with identified pathways was highly relevant to neurodegeneration (e.g., senescence and autophagy). There were also three miRNAs (miR-146b-5p, -150-5p, and -342-3p) that were significantly increased in females vs. males, independent of disease state and APOE-e4 status. We then performed a statistical analysis to assess the effect of APOE genotype in AD within each sex and found that APOE-e4 status affects different subsets of CSF EV miRNAs in females vs. males. Together, this study demonstrates the complexity of the biological factors associated with AD risk and the impact on EV miRNAs, which may contribute to AD pathophysiology. (hide) EV-METRIC 75% (98th 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 Cerebrospinal Fluid 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 Ultrafiltration qEV Protein markers EV: CD9/ CD63/ CD81/ Flotillin1/ TSG101/ Annexin V/ Glast/ CD11b/ NCAM-1/ Synaptophysin/ TMEM119 non-EV: Albumin/ APOA1/ APOE Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cerebrospinal Fluid Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit qEV (35 nm) Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81/ Flotillin1/ TSG101/ Annexin V/ Glast/ CD11b Not detected EV-associated proteins NCAM-1/ Synaptophysin/ TMEM119 Detected contaminants APOE Not detected contaminants Albumin/ APOA1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 76-620 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.50E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-100

	EV220405	3/3	Homo sapiens	Cerebrospinal Fluid	UF qEV	Ursula S Sandau	2022	38%
Study summary Full title Differential Effects of APOE Genotype on MicroRNA Cargo of Cerebrospinal Fluid Extracellular Vesicles in Females With Alzheimer's Disease Compared to Males All authors Ursula S Sandau, Trevor J McFarland, Sierra J Smith, Douglas R Galasko, Joseph F Quinn, Julie A Saugstad Journal Abstract Multiple biological factors, including age, sex, and genetics, influence Alzheimer's disease (AD) ri (show more...)Multiple biological factors, including age, sex, and genetics, influence Alzheimer's disease (AD) risk. Of the 6.2 million Americans living with Alzheimer's dementia in 2021, 3.8 million are women and 2.4 million are men. The strongest genetic risk factor for sporadic AD is apolipoprotein E-e4 (APOE-e4). Female APOE-e4 carriers develop AD more frequently than age-matched males and have more brain atrophy and memory loss. Consequently, biomarkers that are sensitive to biological risk factors may improve AD diagnostics and may provide insight into underlying mechanistic changes that could drive disease progression. Here, we have assessed the effects of sex and APOE-e4 on the miRNA cargo of cerebrospinal fluid (CSF) extracellular vesicles (EVs) in AD. We used ultrafiltration (UF) combined with size exclusion chromatography (SEC) to enrich CSF EVs (e.g., Flotillin+). CSF EVs were isolated from female and male AD or controls (CTLs) that were either APOE-e3,4 or -e3,3 positive (n = 7/group, 56 total). MiRNA expression levels were quantified using a custom TaqMan™ array that assayed 190 miRNAs previously found in CSF, including 25 miRNAs that we previously validated as candidate AD biomarkers. We identified changes in the EV miRNA cargo that were affected by both AD and sex. In total, four miRNAs (miR-16-5p, -331-3p, -409-3p, and -454-3p) were significantly increased in AD vs. CTL, independent of sex and APOE-e4 status. Pathway analysis of the predicted gene targets of these four miRNAs with identified pathways was highly relevant to neurodegeneration (e.g., senescence and autophagy). There were also three miRNAs (miR-146b-5p, -150-5p, and -342-3p) that were significantly increased in females vs. males, independent of disease state and APOE-e4 status. We then performed a statistical analysis to assess the effect of APOE genotype in AD within each sex and found that APOE-e4 status affects different subsets of CSF EV miRNAs in females vs. males. Together, this study demonstrates the complexity of the biological factors associated with AD risk and the impact on EV miRNAs, which may contribute to AD pathophysiology. (hide) EV-METRIC 38% (87th 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 Cerebrospinal Fluid 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 Ultrafiltration qEV Protein markers EV: Flotillin-1/ CD81 non-EV: Albumin/ APOA1 Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cerebrospinal Fluid Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit qEV (70 nm) Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin Not detected EV-associated proteins CD81 Not detected contaminants Albumin/ APOA1 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 86-360 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.54E+09

	EV220405	2/3	Homo sapiens	Cerebrospinal Fluid	UF qEV	Ursula S Sandau	2022	0%
Study summary Full title Differential Effects of APOE Genotype on MicroRNA Cargo of Cerebrospinal Fluid Extracellular Vesicles in Females With Alzheimer's Disease Compared to Males All authors Ursula S Sandau, Trevor J McFarland, Sierra J Smith, Douglas R Galasko, Joseph F Quinn, Julie A Saugstad Journal Abstract Multiple biological factors, including age, sex, and genetics, influence Alzheimer's disease (AD) ri (show more...)Multiple biological factors, including age, sex, and genetics, influence Alzheimer's disease (AD) risk. Of the 6.2 million Americans living with Alzheimer's dementia in 2021, 3.8 million are women and 2.4 million are men. The strongest genetic risk factor for sporadic AD is apolipoprotein E-e4 (APOE-e4). Female APOE-e4 carriers develop AD more frequently than age-matched males and have more brain atrophy and memory loss. Consequently, biomarkers that are sensitive to biological risk factors may improve AD diagnostics and may provide insight into underlying mechanistic changes that could drive disease progression. Here, we have assessed the effects of sex and APOE-e4 on the miRNA cargo of cerebrospinal fluid (CSF) extracellular vesicles (EVs) in AD. We used ultrafiltration (UF) combined with size exclusion chromatography (SEC) to enrich CSF EVs (e.g., Flotillin+). CSF EVs were isolated from female and male AD or controls (CTLs) that were either APOE-e3,4 or -e3,3 positive (n = 7/group, 56 total). MiRNA expression levels were quantified using a custom TaqMan™ array that assayed 190 miRNAs previously found in CSF, including 25 miRNAs that we previously validated as candidate AD biomarkers. We identified changes in the EV miRNA cargo that were affected by both AD and sex. In total, four miRNAs (miR-16-5p, -331-3p, -409-3p, and -454-3p) were significantly increased in AD vs. CTL, independent of sex and APOE-e4 status. Pathway analysis of the predicted gene targets of these four miRNAs with identified pathways was highly relevant to neurodegeneration (e.g., senescence and autophagy). There were also three miRNAs (miR-146b-5p, -150-5p, and -342-3p) that were significantly increased in females vs. males, independent of disease state and APOE-e4 status. We then performed a statistical analysis to assess the effect of APOE genotype in AD within each sex and found that APOE-e4 status affects different subsets of CSF EV miRNAs in females vs. males. Together, this study demonstrates the complexity of the biological factors associated with AD risk and the impact on EV miRNAs, which may contribute to AD pathophysiology. (hide) EV-METRIC 0% (median: 0% 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 Cerebrospinal Fluid Sample origin Alzheimer'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 Ultrafiltration qEV Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cerebrospinal Fluid Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit qEV (35 nm) Other Name other separation method qEV Characterization: Protein analysis None Protein Concentration Method Fluorometric assay Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

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EV-TRACK ID	EV220405
species	Homo sapiens
sample type	Cerebrospinal Fluid
condition	Control condition	Control condition	Alzheimer's disease
separation protocol	Ultrafiltration/ qEV	Ultrafiltration/ qEV	Ultrafiltration/ qEV
Exp. nr.	1	3	2
EV-METRIC %	75	38	0