Application Note

Detection of nucleic acid cargo in EVs by using Fluorescence Nanoparticle Tracking Analysis

Abstract

While the characterization of extracellular vesicles (EVs) was mainly focused on proteins and lipids in the past [1-2], it is apparent that interest in the study of nucleic acid cargo in EVs is now increasing. The use of fluorescence nanoparticle tracking analysis (F-NTA) enables the detection of membrane-associated nucleic acids from EVs.

Keywords: nucleic acid cargo, EVs, F-NTA, fluorescence nanoparticle tracking analysis

EV-membrane-associated nucleic acids labeled with SYBR™ Gold.
Figure 1.: EV-membrane-associated nucleic acids labeled with SYBR™ Gold.

These experiments used the nucleic acid dye SYBR™ Gold (#S11494, Thermo Fisher Scientific), which has shown a sensitivity of >10x more than ethidium bromide, for the detection of DNA and RNA in denaturing urea, glyoxal and formaldehyde gel [3].

The specificity of the experiment was achieved by a nuclease control aimed at specifically degrading EV-associated nucleic acids after labeling them. However, the use of Benzonase (#70746-3, Novagen) did not allow differentiation between DNA and RNA degradation in this experiment.

Particle size distribution of SYBR™ Gold labelled EVs incubated with and without a nuclease prior NTA measurement.
Figure 2.: Particle size distribution of SYBR™ Gold labelled EVs incubated with and without a nuclease prior NTA measurement.
Summary of parameters and results of SYBR™ Gold labeled EVs incubated with and without nuclease.
Table 1: Summary of parameters and results of SYBR™ Gold labeled EVs incubated with and without nuclease.

Conclusions:

The reduction of the fluorescent signal after incubation with Benzonase shows that SYBR™ Gold can be used as a specific stain for nucleic acid detection by F-NTA.

It can be assumed that the SYBR™-Gold signal remaining after nuclease treatment is due to nucleic acids in the EV lumen that cannot be degraded.

Therefore, SYBR™-Gold-activated nucleic acid detection can be used for selective measurement of membrane-associated nucleic acids due to the nuclease treatment.

References

1. Lötvall et al.:
Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles
J Extracell Vesicles 2014; Dec 22:3:26913

2. Théry et al.:
Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines
J Extracell Vesicles 2018; Nov 23;7(1):1535750

3. Truma et al.:
Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators
Anal Biochem 1999; Mar 15;268(2):278-88

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Copyright: © May 2024 Particle Metrix. This publication or parts thereof may not be copied or distributed without our express written permission..

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