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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140065	1/2	Homo sapiens	Serum	(d)(U)C	Cheng L	2014	33%
Study summary Full title Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood All authors Cheng L, Sharples RA, Scicluna BJ, Hill AF Journal J Extracell Vesicles Abstract INTRODUCTION: microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed (show more...)INTRODUCTION: microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed in the host cell and released extracellularly into the bloodstream. Normally involved in post-transcriptional gene silencing, the deregulation of miRNA has been shown to influence pathogenesis of a number of diseases. BACKGROUND: Next-generation deep sequencing (NGS) has provided the ability to profile miRNA in biological fluids making this approach a viable screening tool to detect miRNA biomarkers. However, collection and handling procedures of blood needs to be greatly improved for miRNA analysis in order to reliably detect differences between healthy and disease patients. Furthermore, ribonucleases present in blood can degrade RNA upon collection rendering extracellular miRNA at risk of degradation. These factors have consequently decreased sensitivity and specificity of miRNA biomarker assays. METHODS: Here, we use NGS to profile miRNA in various blood components and identify differences in profiles within peripheral blood compared to cell-free plasma or serum and extracellular vesicles known as exosomes. We also analyse and compare the miRNA content in exosomes prepared by ultracentrifugation methods and commercial exosome isolation kits including treating samples with RNaseA. CONCLUSION: This study demonstrates that exosomal RNA is protected by RNaseA treatment and that exosomes provide a consistent source of miRNA for disease biomarker detection. (hide) EV-METRIC 33% (76th 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 Serum Sample origin NAY Focus vesicles exosomes 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 (d)(U)C Adj. k-factor 122.2 (pelleting) Protein markers EV: TF/ CD63/ Flotillin/ PrP/ HSP70 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70.1Ti Pelleting: adjusted k-factor 122.2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ PrP/ TF/ Flotillin ELISA Antibody details provided? No Detected EV-associated proteins PrP/ TF/ Flotillin Characterization: Particle analysis TRPS EM EM-type transmission EM

	EV140065	2/2	Homo sapiens	Blood plasma	(d)(U)C	Cheng L	2014	33%
Study summary Full title Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood All authors Cheng L, Sharples RA, Scicluna BJ, Hill AF Journal J Extracell Vesicles Abstract INTRODUCTION: microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed (show more...)INTRODUCTION: microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed in the host cell and released extracellularly into the bloodstream. Normally involved in post-transcriptional gene silencing, the deregulation of miRNA has been shown to influence pathogenesis of a number of diseases. BACKGROUND: Next-generation deep sequencing (NGS) has provided the ability to profile miRNA in biological fluids making this approach a viable screening tool to detect miRNA biomarkers. However, collection and handling procedures of blood needs to be greatly improved for miRNA analysis in order to reliably detect differences between healthy and disease patients. Furthermore, ribonucleases present in blood can degrade RNA upon collection rendering extracellular miRNA at risk of degradation. These factors have consequently decreased sensitivity and specificity of miRNA biomarker assays. METHODS: Here, we use NGS to profile miRNA in various blood components and identify differences in profiles within peripheral blood compared to cell-free plasma or serum and extracellular vesicles known as exosomes. We also analyse and compare the miRNA content in exosomes prepared by ultracentrifugation methods and commercial exosome isolation kits including treating samples with RNaseA. CONCLUSION: This study demonstrates that exosomal RNA is protected by RNaseA treatment and that exosomes provide a consistent source of miRNA for disease biomarker detection. (hide) EV-METRIC 33% (65th 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 NAY Focus vesicles exosomes 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 (d)(U)C Adj. k-factor 122.2 (pelleting) Protein markers EV: TF/ CD63/ Flotillin/ PrP/ HSP70 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70.1Ti Pelleting: adjusted k-factor 122.2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ PrP/ TF/ Flotillin ELISA Antibody details provided? No Detected EV-associated proteins PrP/ TF/ Flotillin Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV140065
species	Homo sapiens
sample type	Serum	Blood plasma
condition	NAY	NAY
separation protocol	(d)(U)C	(d)(U)C
Exp. nr.	1	2
EV-METRIC %	33	33