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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140036	1/2	Homo sapiens	Urine	(d)(U)C Filtration	Neeb A	2014	66%
Study summary Full title Splice variant transcripts of the anterior gradient 2 gene as a marker of prostate cancer. All authors Neeb A, Hefele S, Bormann S, Parson W, Adams F, Wolf P, Miernik A, Schoenthaler M, Kroenig M, Wilhelm K, Schultze-Seemann W, Nestel S, Schaefer G, Bu H, Klocker H, Nazarenko I, Cato AC Journal J Cell Sci Abstract Anterior gradient 2 (AGR2) is a gene predominantly expressed in mucus-secreting tissues or in endocr (show more...)Anterior gradient 2 (AGR2) is a gene predominantly expressed in mucus-secreting tissues or in endocrine cells. Its expression is drastically increased in tumors including prostate cancer. Here we investigated whether AGR2 transcript levels can be used as a biomarker to detect prostate cancer (PCa). Using a PCR-based approach, we could show that in addition to the wild-type (AGRwt long and short) transcripts, five other AGR2 splice variants (SV) (referred to as AGR2 SV-C, -E, -F, -G and -H) were present in cancer cell lines. In tissue biopsies, SV-H and AGR2wt (short) distinguished between benign and PCa (p ≤ 0.05 n = 32). In urine exosomes, AGR2 SV-G and SV-H outperformed serum PSA. Receiver operating characteristic (ROC) curves showed the highest discriminatory power of SV-G and SV-H in predicting PCa. AGR2 SV-G and SV-H are potential diagnostic biomarkers for the non-invasive detection of PCa using urine exosomes. (hide) EV-METRIC 66% (93rd 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 Urine Sample origin Prostate cancer 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 (d)(U)C Filtration Adj. k-factor 213.2 (pelleting) Protein markers EV: TSG101/ HSP70/ GAPDH/ CD9/ PSMA non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine 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) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 10-20 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, HSP70, TSG101, PSMA, GAPDH Not detected contaminants Calnexin Characterization: RNA analysis Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 74.0±85.9 EM EM-type Transmission-EM Image type Wide-field

	EV140036	2/2	Homo sapiens	Urine	(d)(U)C Filtration	Neeb A	2014	14%
Study summary Full title Splice variant transcripts of the anterior gradient 2 gene as a marker of prostate cancer. All authors Neeb A, Hefele S, Bormann S, Parson W, Adams F, Wolf P, Miernik A, Schoenthaler M, Kroenig M, Wilhelm K, Schultze-Seemann W, Nestel S, Schaefer G, Bu H, Klocker H, Nazarenko I, Cato AC Journal J Cell Sci Abstract Anterior gradient 2 (AGR2) is a gene predominantly expressed in mucus-secreting tissues or in endocr (show more...)Anterior gradient 2 (AGR2) is a gene predominantly expressed in mucus-secreting tissues or in endocrine cells. Its expression is drastically increased in tumors including prostate cancer. Here we investigated whether AGR2 transcript levels can be used as a biomarker to detect prostate cancer (PCa). Using a PCR-based approach, we could show that in addition to the wild-type (AGRwt long and short) transcripts, five other AGR2 splice variants (SV) (referred to as AGR2 SV-C, -E, -F, -G and -H) were present in cancer cell lines. In tissue biopsies, SV-H and AGR2wt (short) distinguished between benign and PCa (p ≤ 0.05 n = 32). In urine exosomes, AGR2 SV-G and SV-H outperformed serum PSA. Receiver operating characteristic (ROC) curves showed the highest discriminatory power of SV-G and SV-H in predicting PCa. AGR2 SV-G and SV-H are potential diagnostic biomarkers for the non-invasive detection of PCa using urine exosomes. (hide) EV-METRIC 14% (40th 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 Urine Sample origin Benign prostate hyperplasia 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 (d)(U)C Filtration Adj. k-factor 213.2 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine 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) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method microBCA Protein Yield (µg) 10-20 Characterization: RNA analysis Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV140036
species	Homo sapiens
sample type	Urine
condition	Prostate cancer	Benign prostate hyperplasia
separation protocol	(d)(U)C Filtration	(d)(U)C Filtration
Exp. nr.	1	2
EV-METRIC %	66	14