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You searched for: EV200031 (EV-TRACK ID)

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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
EV200031 4/8 Homo sapiens Blood plasma DG
(d)(U)C
Grossi, Ilaria 2020 89%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
89% (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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
EV density (g/ml)
1.09-1.22
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
7
Lowest density fraction
15%
Highest density fraction
70%
Total gradient volume, incl. sample (mL)
4.4
Sample volume (mL)
1
Orientation
Top-down
Rotor type
MLS-50
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
0.4
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
50-100
Report type
Not Reported
EV200031 8/8 Homo sapiens Blood plasma DG
(d)(U)C
Grossi, Ilaria 2020 89%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
89% (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
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
Parkinson's disease
Focus vesicles
extracellular vesicle
Separation protocol
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
DG
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
EV density (g/ml)
1.09-1.22
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Parkinson's disease
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
7
Lowest density fraction
15%
Highest density fraction
70%
Total gradient volume, incl. sample (mL)
4.4
Sample volume (mL)
1
Orientation
Top-down
Rotor type
MLS-50
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
0.4
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
50-100
Report type
Not Reported
EV200031 1/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th 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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
30
Pelleting: rotor type
45-30-11 rotor Eppendorf
Pelleting: speed (g)
800
Wash: volume per pellet (ml)
1
Wash: time (min)
30
Wash: Rotor Type
45-30-11 rotor eppendorf
Wash: speed (g)
800
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
200-600
Report type
Not Reported
EV200031 2/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th 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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
45
Pelleting: rotor type
45-30-11 rotor Eppendorf
Pelleting: speed (g)
16000
Wash: volume per pellet (ml)
1
Wash: time (min)
45
Wash: Rotor Type
45-30-11 rotor eppendorf
Wash: speed (g)
16000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
50-400
Report type
Not Reported
EV200031 3/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th 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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
30-200
Report type
Not Reported
EV200031 5/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th 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
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
Parkinson's disease
Focus vesicles
extracellular vesicle
Separation protocol
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
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Parkinson's disease
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
30
Pelleting: rotor type
45-30-11 rotor Eppendorf
Pelleting: speed (g)
800
Wash: volume per pellet (ml)
1
Wash: time (min)
30
Wash: Rotor Type
45-30-11 rotor eppendorf
Wash: speed (g)
800
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
200-600
Report type
Not Reported
EV200031 6/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th 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
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
Parkinson's disease
Focus vesicles
extracellular vesicle
Separation protocol
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
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Parkinson's disease
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
45
Pelleting: rotor type
45-30-11 rotor Eppendorf
Pelleting: speed (g)
16000
Wash: volume per pellet (ml)
1
Wash: time (min)
45
Wash: Rotor Type
45-30-11 rotor eppendorf
Wash: speed (g)
16000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
50-400
Report type
Not Reported
EV200031 7/8 Homo sapiens Blood plasma (d)(U)C Grossi, Ilaria 2020 67%

Study summary

Full title
All authors
Ilaria Grossi, Annalisa Radeghieri, Lucia Paolini, Vanessa Porrini, Andrea Pilotto, Alessandro Padovani, Alessandra Marengoni, Alessandro Barbon, Arianna Bellucci, Marina Pizzi, Alessandro Salvi, Giuseppina De Petro
Journal
Int J Mol Med
Abstract
Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most com (show more...)Parkinson's disease (PD) is an important disabling age‑related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle‑free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood‑based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR‑34a‑5p because of its well‑documented involvement in neurobiology. To explore a differential profile of circulating miR‑34a‑5p in PD, PD patients and age‑matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR‑34a‑5p levels were evaluated using qPCR in plasma and in each EV type. miR‑34a‑5p was significantly up‑regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR‑34a‑5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR‑34a‑5p in PD via the use of pure SEVs. (hide)
EV-METRIC
67% (95th 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
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
Parkinson's disease
Focus vesicles
extracellular vesicle
Separation protocol
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
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Annexin-V/ Flotillin1/ Adam-10/ Actinin-4
non-EV: Apo-AI/ GM130
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Parkinson's disease
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Adam-10/ Actinin-4/ Annexin-V/ TSG101/ Alix/ CD81
Not detected contaminants
Apo-AI/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase H
RNAse concentration
0.00625
Characterization: Particle analysis
EM
EM-type
Atomic force microscopy
Image type
Close-up, Wide-field
Report size (nm)
30-200
Report type
Not Reported
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