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Results list Study Tree
Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV190044 11/11 Homo sapiens Blood plasma DG
(d)(U)C
qEV 		Driedonks, Tom A.P. 2020 100%

Study summary

Full title
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
100% (99th 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
DG
(d)(U)C
qEV
Protein markers
EV: CD81/ CD63/ Flotillin1/ CD9
non-EV: ApoAl/ ApoB100
Proteomics
no
EV density (g/ml)
1.11 - 1.18
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.05
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Commercial kit
qEV
Other
Name other separation method
qEV
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
Flotillin1/ CD9/ CD63/ CD81
Not detected contaminants
ApoAl/ ApoB100
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
0.045
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.16
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
130
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190044 5/11 Homo sapiens primary neutrophils DG
(d)(U)C 		Driedonks, Tom A.P. 2020 67%

Study summary

Full title
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
67% (94th 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
DG
(d)(U)C
Protein markers
EV: CD63/ CD9
non-EV: Calnexin
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary neutrophils
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63
Not detected contaminants
Calnexin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012.
Calibration bead size
0.1
EV concentration
Yes
EV190044 6/11 Homo sapiens primary neutrophils DG
(d)(U)C 		Driedonks, Tom A.P. 2020 67%

Study summary

Full title
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
67% (94th 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
LPS stimulated
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
DG
(d)(U)C
Protein markers
EV: CD63/ CD9
non-EV: Calnexin
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary neutrophils
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63
Not detected contaminants
Calnexin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012.
Calibration bead size
0.1
EV190044 1/11 Homo sapiens HUVEC DG
(d)(U)C 		Driedonks, Tom A.P. 2020 43%

Study summary

Full title
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
43% (81st 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
DG
(d)(U)C
Protein markers
EV:
non-EV:
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HUVEC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012. We applied fluorescence threshold triggering to discriminate PKH67 labeled EV from non-fluorescent noise signals. Forward scatter (FSC) was detected at a 15 25 degree collection angle. Fluorescent polystyrene 100 and 200 nm beads (FluoSpheres, Invitrogen, Carlsbad, CA) were used to calibrate the fluorescence and reduced width-FSC settings before each measurement. Samples were measured at maximally 10,000 events per second, which is far below the electronic pulse processing limit of the BD Influx. Serial dilutions of 'peak' fractions were included to control for potential 'invisible swarm' effects
Calibration bead size
0.1
EV concentration
Yes
EV190044 2/11 Homo sapiens HUVEC DG
(d)(U)C 		Driedonks, Tom A.P. 2020 43%

Study summary

Full title
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
43% (81st 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
LPS stimulated
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
DG
(d)(U)C
Protein markers
EV:
non-EV:
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HUVEC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012.
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EV190044 3/11 Homo sapiens primary PBMC DG
(d)(U)C 		Driedonks, Tom A.P. 2020 43%

Study summary

Full title
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
43% (81st 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
DG
(d)(U)C
Protein markers
EV:
non-EV:
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary PBMC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012.
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EV190044 4/11 Homo sapiens primary PBMC DG
(d)(U)C 		Driedonks, Tom A.P. 2020 43%

Study summary

Full title
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
43% (81st 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
LPS stimulated
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
DG
(d)(U)C
Protein markers
EV:
non-EV:
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary PBMC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012.
Calibration bead size
0.1
Report type
Not Reported
EV190044 7/11 Homo sapiens red blood cells DG
(d)(U)C 		Driedonks, Tom A.P. 2020 43%

Study summary

Full title
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
43% (81st 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
DG
(d)(U)C
Protein markers
EV:
non-EV:
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
red blood cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012.
Calibration bead size
0.1
EV concentration
Yes
EV190044 8/11 Homo sapiens red blood cells DG
(d)(U)C 		Driedonks, Tom A.P. 2020 43%

Study summary

Full title
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
43% (81st 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
LPS stimulated
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
DG
(d)(U)C
Protein markers
EV:
non-EV:
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
red blood cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012.
Calibration bead size
0.1
EV190044 9/11 Homo sapiens platelets DG
(d)(U)C 		Driedonks, Tom A.P. 2020 43%

Study summary

Full title
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
43% (81st 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
DG
(d)(U)C
Protein markers
EV:
non-EV:
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
platelets
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012.
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EV190044 10/11 Homo sapiens platelets DG
(d)(U)C 		Driedonks, Tom A.P. 2020 43%

Study summary

Full title
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
43% (81st 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
LPS stimulated
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
DG
(d)(U)C
Protein markers
EV:
non-EV:
Proteomics
no
EV density (g/ml)
1.10 - 1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
platelets
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.02
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
Capillary electrophoresis (e.g. Bioanalyzer);(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
See van der Vlist, Nature Protocols 2012 and Nolte-'t Hoen, Nanomedicine 2012.
Calibration bead size
0.1
Report type
Not Reported
1 - 11 of 11
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV190044
species
Homo
sapiens
sample type
Blood
plasma
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
cell type
NA
primary
neutrophils
primary
neutrophils
HUVEC
HUVEC
primary
PBMC
primary
PBMC
red
blood
cells
red
blood
cells
platelets
platelets
medium
NA
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
condition
Control
condition
Control
condition
LPS
stimulated
Control
condition
LPS
stimulated
Control
condition
LPS
stimulated
Control
condition
LPS
stimulated
Control
condition
LPS
stimulated
separation protocol
DG
(d)(U)C
qEV
DG
(d)(U)C
DG
(d)(U)C
DG
(d)(U)C
DG
(d)(U)C
DG
(d)(U)C
DG
(d)(U)C
DG
(d)(U)C
DG
(d)(U)C
DG
(d)(U)C
DG
(d)(U)C
Exp. nr.
11
5
6
1
2
3
4
7
8
9
10
EV-METRIC %
100
67
67
43
43
43
43
43
43
43
43