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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
EV190037 1/1 Homo sapiens Blood plasma Filtration
qEV
Laetitia S. Gaspar 2020 75%

Study summary

Full title
All authors
Laetitia S. Gaspar, Magda M. Santana, Carina Henriques, Maria M. Pinto, Teresa M. Ribeiro-Rodrigues, Henrique Girão, Rui Jorge Nobre, Luís Pereira de Almeida
Journal
Methods & Clinical Development
Abstract
Extracellular vesicles (EVs) are membranous structures that protect RNAs from damage when circulatin (show more...)Extracellular vesicles (EVs) are membranous structures that protect RNAs from damage when circulating in complex biological fluids, such as plasma. RNAs are extremely specific to health and disease, being powerful tools for diagnosis, treatment response monitoring, and development of new therapeutic strategies for several diseases. In this context, EVs are potential sources of disease biomarkers and promising delivery vehicles. However, standardized and reproducible EV isolation protocols easy to implement in clinical practice are missing. Here, a size exclusion chromatography-based protocol for EV-isolation from human plasma was optimized. We propose a workflow to isolate EVs for transcriptional research that allows concomitant analysis of particle number and size, total protein, and quantification of a major plasma contaminant. This protocol yields 7.54 × 109 ± 1.22 × 108 particles, quantified by nanoparticle tracking analysis, with a mean size of 115.7 ± 11.12 nm and a mode size of 83.13 ± 4.72 nm, in a ratio of 1.19 × 1010 ± 7.38 × 109 particles/μg of protein, determined by Micro Bicinchoninic Acid (BCA) Protein Assay, and 3.09 ± 0.7 ng RNA, assessed by fluorescence-based RNA-quantitation, from only 900 μL of plasma. The protocol is fast and easy to implement and has potential for application in biomarkers research, therapeutic strategies development, and clinical practice. (hide)
EV-METRIC
75% (96th 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
Filtration
qEV
Protein markers
EV: TSG101/ Analysed only by WB and Dot Blot. We used ELISA to analyse the presence of contaminants/ CD63/ CD81/ GAPDH/ Anxa5/ Alix/ ICAM/ Flotillin1/ LAMP2/ EpCAM
non-EV: Calnexin/ Albumin/ GM130/ ApoB100/ ApoA1
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Filtration steps
> 0.45 µm,
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ LAMP2/ CD81
Not detected EV-associated proteins
GAPDH
Not detected contaminants
Calnexin
ELISA
Antibody details provided?
No
Detected EV-associated proteins
Analysed only by WB and Dot Blot. We used ELISA to analyse the presence of contaminants
Detected contaminants
ApoA1/ ApoB100/ Albumin
Other 1
Dot Blot Array
Detected EV-associated proteins
Flotillin1/ CD63/ EpCAM/ ICAM/ Anxa5/ CD81/ TSG101
Not detected EV-associated proteins
Alix
Not detected contaminants
GM130
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
NTA
Report type
Mean
Reported size (nm)
89.04
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
1 - 1 of 1
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV190037
species
Homo sapiens
sample type
Blood plasma
condition
Control condition
separation protocol
Filtration
qEV
Exp. nr.
1
EV-METRIC %
75