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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100066	2/2	Bos bovis	Milk	(d)(U)C DG	Hata T	2010	33%
Study summary Full title Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs All authors Hata T, Murakami K, Nakatani H, Yamamoto Y, Matsuda T, Aoki N Journal Biochem Biophys Res Commun Abstract By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approxima (show more...)By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approximately 100 nm in diameter from bovine milk. We also found that approximately 1700 and 1000 ng of total RNA, in which small RNAs were major components, was contained inside the microvesicles isolated from 6 ml of colostrum and mature milk, respectively, despite high RNase activity in the milk. Polyadenylated gene transcripts for major milk proteins and translation elongation factor-1alpha (EF-1alpha) were present in the microvesicles, and integrity of some transcripts was confirmed by real-time PCR targeting 5'- and 3'-ends of mRNA and by in vitro translation analysis. Moreover, a considerable amount of mammary gland and immune-related microRNAs were present in the milk-derived microvesicles. Acidification of milk to mimic gastrointestinal tract did not mostly affected RNA yield and quality. The milk related gene transcripts were detected in cultured cells when incubated with milk-derived microvesicles, suggesting cellular uptake of the microvesicle contents including RNA. Our findings suggest that bovine breast milk contains RNAs capable for being transferred to living cells and involved in the development of calf's gastrointestinal and immune systems. (hide) EV-METRIC 33% (53rd 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 Milk Sample origin NAY Focus vesicles microvesicles 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 DG Adj. k-factor 255.8 (pelleting) Protein markers EV: MFGE8 non-EV: Proteomics no EV density (g/ml) 1.16-1.2 Show all info Study aim Function Sample Species Bos bovis Sample Type Milk 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Density gradient Only used for validation of main results Yes Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins MFGE8 ELISA Antibody details provided? No Detected EV-associated proteins MFGE8 Characterization: Particle analysis EM EM-type transmission EM/ scanning EM Image type Close-up Report size (nm) Not reported

	EV100066	1/2	Bos bovis	Milk	(d)(U)C	Hata T	2010	14%
Study summary Full title Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs All authors Hata T, Murakami K, Nakatani H, Yamamoto Y, Matsuda T, Aoki N Journal Biochem Biophys Res Commun Abstract By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approxima (show more...)By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approximately 100 nm in diameter from bovine milk. We also found that approximately 1700 and 1000 ng of total RNA, in which small RNAs were major components, was contained inside the microvesicles isolated from 6 ml of colostrum and mature milk, respectively, despite high RNase activity in the milk. Polyadenylated gene transcripts for major milk proteins and translation elongation factor-1alpha (EF-1alpha) were present in the microvesicles, and integrity of some transcripts was confirmed by real-time PCR targeting 5'- and 3'-ends of mRNA and by in vitro translation analysis. Moreover, a considerable amount of mammary gland and immune-related microRNAs were present in the milk-derived microvesicles. Acidification of milk to mimic gastrointestinal tract did not mostly affected RNA yield and quality. The milk related gene transcripts were detected in cultured cells when incubated with milk-derived microvesicles, suggesting cellular uptake of the microvesicle contents including RNA. Our findings suggest that bovine breast milk contains RNAs capable for being transferred to living cells and involved in the development of calf's gastrointestinal and immune systems. (hide) EV-METRIC 14% (21st 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 Milk Sample origin NAY Focus vesicles microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 255.8 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Bos bovis Sample Type Milk 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Characterization: Particle analysis None

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EV-TRACK ID	EV100066
species	Bos bovis
sample type	Milk
condition	NAY
separation protocol	(d)(U)C DG	(d)(U)C
Exp. nr.	2	1
EV-METRIC %	33	14