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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210501	1/2	Homo sapiens	MCF-7	(d)(U)C	Zhai LY	2018	56%
Study summary Full title In Situ Detection of Plasma Exosomal MicroRNA-1246 for Breast Cancer Diagnostics by a Au Nanoflare Probe. All authors Zhai LY, Li MX, Pan WL, Chen Y, Li MM, Pang JX, Zheng L, Chen JX, Duan WJ Journal ACS Appl Mater Interfaces Abstract Breast cancer is the second cause of cancer mortality in women globally. Early detection, treatment, (show more...)Breast cancer is the second cause of cancer mortality in women globally. Early detection, treatment, and metastasis monitoring are of great importance to favorable prognosis. Although conventional diagnostic methods, such as breast X-ray mammography and image positioning biopsy, are accurate, they could cause radioactive or invasive damage to patients. Liquid biopsy as a noninvasive method is convenient for repeated sampling in clinical cancer prognostic, metastatic evaluation, and relapse monitoring. MicroRNAs encased in exosomes circulating in biofluids are promising candidate cancer biomarkers because of their cancer-specific expression profiles. Here, we report an in situ detection of microRNA-1246 (miR-1246) in human plasma exosomes as breast cancer biomarker by a nucleic acid functionalized Au nanoflare probe. Needing neither time-consuming and costly isolation of exosomes from the plasma sample nor transfection means, the Au nanoflare probe can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting miR-1246. Only 40 μL of plasma is needed to incubate 4 h with the probe, giving signal sensitive enough to distinguish samples of breast cancer to normal control. Using plasma miR-1246 level detected by our assay as a marker, we differentiated 46 breast cancer patients from 28 healthy controls with 100% sensitivity and 92.9% specificity at the best cutoff. This simple, accurate, sensitive, and cost-effective liquid biopsy by the Au nanoflare probe is potent to be developed as a noninvasive breast cancer diagnostic assay for clinical adaption. (hide) EV-METRIC 56% (90th 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 exosome 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 Protein markers EV: TSG101/ CD63 non-EV: None Proteomics no Show all info Study aim New methodological development/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF-7 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: time (min) 80 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 64 RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 4 U / mL Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 97.8 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 67.7

	EV210501	2/2	Homo sapiens	MCF-10A	(d)(U)C	Zhai LY	2018	14%
Study summary Full title In Situ Detection of Plasma Exosomal MicroRNA-1246 for Breast Cancer Diagnostics by a Au Nanoflare Probe. All authors Zhai LY, Li MX, Pan WL, Chen Y, Li MM, Pang JX, Zheng L, Chen JX, Duan WJ Journal ACS Appl Mater Interfaces Abstract Breast cancer is the second cause of cancer mortality in women globally. Early detection, treatment, (show more...)Breast cancer is the second cause of cancer mortality in women globally. Early detection, treatment, and metastasis monitoring are of great importance to favorable prognosis. Although conventional diagnostic methods, such as breast X-ray mammography and image positioning biopsy, are accurate, they could cause radioactive or invasive damage to patients. Liquid biopsy as a noninvasive method is convenient for repeated sampling in clinical cancer prognostic, metastatic evaluation, and relapse monitoring. MicroRNAs encased in exosomes circulating in biofluids are promising candidate cancer biomarkers because of their cancer-specific expression profiles. Here, we report an in situ detection of microRNA-1246 (miR-1246) in human plasma exosomes as breast cancer biomarker by a nucleic acid functionalized Au nanoflare probe. Needing neither time-consuming and costly isolation of exosomes from the plasma sample nor transfection means, the Au nanoflare probe can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting miR-1246. Only 40 μL of plasma is needed to incubate 4 h with the probe, giving signal sensitive enough to distinguish samples of breast cancer to normal control. Using plasma miR-1246 level detected by our assay as a marker, we differentiated 46 breast cancer patients from 28 healthy controls with 100% sensitivity and 92.9% specificity at the best cutoff. This simple, accurate, sensitive, and cost-effective liquid biopsy by the Au nanoflare probe is potent to be developed as a noninvasive breast cancer diagnostic assay for clinical adaption. (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 exosome 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim New methodological development/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF-10A EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: time (min) 80 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 64 RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 4 U / mL Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV210501
species	Homo sapiens
sample type	Cell culture
cell type	MCF-7	MCF-10A
condition	Control condition	Control condition
separation protocol	dUC	dUC
Exp. nr.	1	2
EV-METRIC %	56	14