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
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.
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.
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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