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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200008	1/18	Homo sapiens	Blood plasma	(d)(U)C	Peng, Cheng	2020	56%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 56% (88th 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 (d)(U)C Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 660 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ vimentin/ TSG101/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3070000000

	EV200008	10/18	Homo sapiens	Blood plasma	miRCURY Exosome Kit	Peng, Cheng	2020	56%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 56% (88th 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 miRCURY Exosome Kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;miRCURY Exosome Kit Other Name other separation method miRCURY Exosome Kit Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101/ CD81 Not detected EV-associated proteins vimentin/ CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 56700000000

	EV200008	2/18	Homo sapiens	Blood plasma	Wayen Exosome Isolation kit	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Wayen Exosome Isolation kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Wayen Exosome Isolation kit Other Name other separation method Wayen Exosome Isolation kit Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ vimentin/ TSG101/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 86700000000

	EV200008	3/18	Homo sapiens	Blood plasma	ExoQuick	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 ExoQuick Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ vimentin/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 24300000000

	EV200008	4/18	Homo sapiens	Blood plasma	(d)(U)C	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma 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 (d)(U)C Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 660 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins vimentin/ CD9/ CD63/ TSG101/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 733000000

	EV200008	5/18	Homo sapiens	Blood plasma	Wayen Exosome Isolation kit	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma 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 Wayen Exosome Isolation kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Wayen Exosome Isolation kit Other Name other separation method Wayen Exosome Isolation kit Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins vimentin/ CD9/ CD63/ TSG101/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 90000000000

	EV200008	6/18	Homo sapiens	Blood plasma	ExoQuick	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma 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 ExoQuick Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins vimentin/ TSG101/ CD9/ CD63/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 53300000000

	EV200008	7/18	Homo sapiens	Blood plasma	(d)(U)C	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma 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 (d)(U)C Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 660 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins vimentin/ TSG101/ CD9/ CD63/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6670000000

	EV200008	8/18	Homo sapiens	Blood plasma	Wayen Exosome Isolation kit	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma 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 Wayen Exosome Isolation kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Wayen Exosome Isolation kit Other Name other separation method Wayen Exosome Isolation kit Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins vimentin/ CD9/ CD63/ TSG101/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 227000000000

	EV200008	9/18	Homo sapiens	Blood plasma	ExoQuick	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma 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 ExoQuick Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins vimentin/ TSG101/ CD9/ CD63/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 32000000000

	EV200008	11/18	Homo sapiens	Blood plasma	Ribo Exosome Isolation Reagent	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Ribo Exosome Isolation Reagent Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Ribo Exosome Isolation Reagent Other Name other separation method Ribo Exosome Isolation Reagent Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101/ CD81 Not detected EV-associated proteins vimentin/ CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 86700000000

	EV200008	12/18	Homo sapiens	Blood plasma	Total Exosome Isolation	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Total Exosome Isolation Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81 Not detected EV-associated proteins vimentin/ TSG101/ CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 36700000000

	EV200008	13/18	Homo sapiens	Blood plasma	miRCURY Exosome Kit	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma 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 miRCURY Exosome Kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;miRCURY Exosome Kit Other Name other separation method miRCURY Exosome Kit Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101/ CD81 Not detected EV-associated proteins vimentin/ CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 63300000000

	EV200008	14/18	Homo sapiens	Blood plasma	Ribo Exosome Isolation Reagent	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma 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 Ribo Exosome Isolation Reagent Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Ribo Exosome Isolation Reagent Other Name other separation method Ribo Exosome Isolation Reagent Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins vimentin/ CD63/ TSG101/ CD81 Not detected EV-associated proteins CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 70000000000

	EV200008	15/18	Homo sapiens	Blood plasma	Total Exosome Isolation	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 primary osteosarcoma 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 Total Exosome Isolation Protein markers EV: TSG101/ CD81/ CD63/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins vimentin/ CD63/ CD81 Not detected EV-associated proteins TSG101/ CD63 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 103000000000

	EV200008	16/18	Homo sapiens	Blood plasma	miRCURY Exosome Kit	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma 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 miRCURY Exosome Kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;miRCURY Exosome Kit Other Name other separation method miRCURY Exosome Kit Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins vimentin/ TSG101/ CD63/ CD81 Not detected EV-associated proteins CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 60000000000

	EV200008	17/18	Homo sapiens	Blood plasma	Ribo Exosome Isolation Reagent	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma 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 Ribo Exosome Isolation Reagent Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;Ribo Exosome Isolation Reagent Other Name other separation method Ribo Exosome Isolation Reagent Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins vimentin/ CD63/ TSG101/ CD81 Not detected EV-associated proteins CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 160000000000

	EV200008	18/18	Homo sapiens	Blood plasma	Total Exosome Isolation	Peng, Cheng	2020	50%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 50% (83rd 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 Metastatic osteosarcoma 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 Total Exosome Isolation Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins vimentin/ TSG101/ CD81 Not detected EV-associated proteins CD63/ CD9 Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 107000000000

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EV-TRACK ID	EV200008
species	Homo sapiens
sample type	Blood plasma
condition	Control condition	Control condition	Control condition	Control condition	primary osteosarcoma	primary osteosarcoma	primary osteosarcoma	Metastatic osteosarcoma	Metastatic osteosarcoma	Metastatic osteosarcoma	Control condition	Control condition	primary osteosarcoma	primary osteosarcoma	primary osteosarcoma	Metastatic osteosarcoma	Metastatic osteosarcoma	Metastatic osteosarcoma
separation protocol	(d)(U)C	miRCURY Exosome Kit	Wayen Exosome Isolation kit	ExoQuick	(d)(U)C	Wayen Exosome Isolation kit	ExoQuick	(d)(U)C	Wayen Exosome Isolation kit	ExoQuick	Ribo Exosome Isolation Reagent	Total Exosome Isolation	miRCURY Exosome Kit	Ribo Exosome Isolation Reagent	Total Exosome Isolation	miRCURY Exosome Kit	Ribo Exosome Isolation Reagent	Total Exosome Isolation
Exp. nr.	1	10	2	3	4	5	6	7	8	9	11	12	13	14	15	16	17	18
EV-METRIC %	56	56	50	50	50	50	50	50	50	50	50	50	50	50	50	50	50	50

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