Related Experiment Video
Updated: Jun 1, 2026
Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media
Published on: June 14, 2013
Characterization of extracellular circulating microRNA
Andrey Turchinovich1, Ludmila Weiz, Anne Langheinz
1Molecular Epidemiology Group, German Cancer Research Center, Heidelberg, Germany. a.turchinovich@dkfz.de
Abstract:
MicroRNAs (miRNAs), a class of post-transcriptional gene expression regulators, have recently been detected in human body fluids, including peripheral blood plasma as extracellular nuclease resistant entities. However, the origin and function of extracellular circulating miRNA remain essentially unknown. Here, we confirmed that circulating mature miRNA in contrast to mRNA or snRNA is strikingly stable in blood plasma and cell culture media. Furthermore, we found that most miRNA in plasma and cell culture media completely passed through 0.22 µm filters but remained in the supernatant after ultracentrifugation at 110 000g indicating the non-vesicular origin of the extracellular miRNA. Furthermore, western blot immunoassay revealed that extracellular miRNA ultrafiltrated together with the 96 kDa Ago2 protein, a part of RNA-induced silencing complex. Moreover, miRNAs in both blood plasma and cell culture media co-immunoprecipited with anti-Ago2 antibody in a detergent free environment. This is the first study to show that extracellular miRNAs are predominantly exosomes/microvesicles free and are associated with Ago proteins. We hypothesize that extracellular miRNAs are in the most part by-products of dead cells that remain in extracellular space due to the high stability of the Ago2 protein and Ago2-miRNA complex. Nevertheless, our data does not reject the possibility that some miRNAs can be associated with exosomes.
Insights
Extracellular microRNAs (miRNAs) are stable in body fluids and primarily associate with Ago2 proteins, not vesicles. This suggests they originate from dead cells, offering new insights into circulating miRNA function.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- MicroRNAs (miRNAs) are key post-transcriptional gene regulators.
- Extracellular miRNAs are found in human body fluids but their origin and function are unclear.
- Circulating miRNAs are notably stable and resistant to nucleases.
Purpose of the Study:
- To investigate the origin and characteristics of extracellular circulating miRNAs.
- To determine if extracellular miRNAs are vesicle-associated or free in plasma.
- To elucidate the protein interactions of extracellular miRNAs.
Main Methods:
- Assessed miRNA stability in plasma and cell culture media.
- Utilized 0.22 µm filtration and ultracentrifugation to analyze miRNA localization.
- Performed western blot and co-immunoprecipitation assays with anti-Ago2 antibody.
Main Results:
- Circulating mature miRNAs are highly stable in plasma, unlike mRNA or snRNA.
- Most extracellular miRNAs passed through filters and remained in supernatant after ultracentrifugation, indicating non-vesicular origin.
- Extracellular miRNAs were found to be associated with Ago2 protein and co-immunoprecipitated with anti-Ago2 antibodies.
Conclusions:
- Extracellular miRNAs are predominantly free of exosomes/microvesicles and associated with Ago2 proteins.
- The stability of the Ago2-miRNA complex likely contributes to extracellular miRNA persistence.
- Extracellular miRNAs may originate as by-products of dead cells, though exosome association cannot be entirely excluded.
Related Concept Videos
01:22MicroRNAs
01:22MicroRNAs
01:22MicroRNAs