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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210320	2/2	Homo sapiens	HEK293T	(d)(U)C UF	Lamichhane TN	2016	33%
Study summary Full title Oncogene Knockdown via Active Loading of Small RNAs into Extracellular Vesicles by Sonication. All authors Lamichhane TN, Jeyaram A, Patel DB, Parajuli B, Livingston NK, Arumugasaamy N, Schardt JS, Jay SM Journal Cell Mol Bioeng Abstract Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug d (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug delivery vehicles for small RNAs (siRNA and miRNA) due to their natural role in intercellular RNA transport. However, the application of EVs for therapeutic RNA delivery may be limited by loading approaches that can induce cargo aggregation or degradation. Here, we report the use of sonication as a means to actively load functional small RNAs into EVs. Conditions under which EVs could be loaded with small RNAs with minimal detectable aggregation were identified, and EVs loaded with therapeutic siRNA via sonication were observed to be taken up by recipient cells and capable of target mRNA knockdown leading to reduced protein expression. This system was ultimately applied to reduce expression of HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, and could be extended to other therapeutic targets. These results define important parameters informing the application of sonication as a small RNA loading method for EVs and demonstrate the potential utility of this approach for versatile cancer therapy. (hide) EV-METRIC 33% (75th 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 Cell culture supernatant 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 (Differential) (ultra)centrifugation Ultrafiltration Protein markers EV: Alix/ TSG101 non-EV: GAPDH Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium EV-depleted medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 300 Membrane type NS Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ TSG101 Detected contaminants GAPDH Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 134 EV concentration Yes

	EV210320	1/2	Homo sapiens	MCF7	(d)(U)C UF	Lamichhane TN	2016	14%
Study summary Full title Oncogene Knockdown via Active Loading of Small RNAs into Extracellular Vesicles by Sonication. All authors Lamichhane TN, Jeyaram A, Patel DB, Parajuli B, Livingston NK, Arumugasaamy N, Schardt JS, Jay SM Journal Cell Mol Bioeng Abstract Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug d (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug delivery vehicles for small RNAs (siRNA and miRNA) due to their natural role in intercellular RNA transport. However, the application of EVs for therapeutic RNA delivery may be limited by loading approaches that can induce cargo aggregation or degradation. Here, we report the use of sonication as a means to actively load functional small RNAs into EVs. Conditions under which EVs could be loaded with small RNAs with minimal detectable aggregation were identified, and EVs loaded with therapeutic siRNA via sonication were observed to be taken up by recipient cells and capable of target mRNA knockdown leading to reduced protein expression. This system was ultimately applied to reduce expression of HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, and could be extended to other therapeutic targets. These results define important parameters informing the application of sonication as a small RNA loading method for EVs and demonstrate the potential utility of this approach for versatile cancer therapy. (hide) EV-METRIC 14% (44th 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 Cell culture supernatant 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 (Differential) (ultra)centrifugation Ultrafiltration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium EV-depleted medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 300 Membrane type NS Characterization: Protein analysis None Protein Concentration Method BCA Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV210320
species	Homo sapiens
sample type	Cell culture
cell type	HEK293T	MCF7
condition	Control condition	Control condition
separation protocol	dUC/ Ultrafiltration	dUC/ Ultrafiltration
Exp. nr.	2	1
EV-METRIC %	33	14