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MicroRNAs01:22

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MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After...
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Related Experiment Video

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Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells
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Extracellular microRNAs exhibit sequence-dependent stability and cellular release kinetics.

Anna M L Coenen-Stass1, Marie J Pauwels1,2,3, Britt Hanson1,4

  • 1a Department of Physiology, Anatomy and Genetics , University of Oxford , Oxford , UK.

RNA Biology
|March 7, 2019
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Summary

Extracellular microRNAs (ex-miRNAs) are rapidly degraded at body temperature, challenging their use as biomarkers. However, ex-miRNAs within extracellular vesicles remain stable, suggesting differential stability mechanisms.

Keywords:
Extracellular microRNAex-miRNAhalf-lifekineticsmiRNAmicroRNAserum

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Extracellular microRNAs (ex-miRNAs) are considered stable in extracellular environments at low temperatures.
  • Previous studies suggest ex-miRNAs are robust biomarkers due to their stability.

Purpose of the Study:

  • To investigate the stability of ex-miRNAs under physiological conditions (37°C with serum).
  • To determine the factors influencing ex-miRNA stability and release kinetics.
  • To assess the implications for ex-miRNA utility as biomarkers and in intercellular communication.

Main Methods:

  • Incubation of ex-miRNAs at 37°C with serum to simulate physiological conditions.
  • Measurement of ex-miRNA half-lives in different biofluids (murine serum, conditioned medium).
  • Isolation of ex-miRNAs from extracellular vesicles using size exclusion chromatography.
  • Time-course measurement of ex-miRNA release from cells.
  • Mathematical modeling of miRNA release and degradation kinetics.

Main Results:

  • Many ex-miRNAs are rapidly degraded at 37°C in serum, with half-lives ranging from ~1.5 to over 13 hours.
  • Ex-miRNA stability in different biofluids was similar, indicating sequence-dependent stability.
  • Ex-miRNAs encapsulated within extracellular vesicles demonstrated high stability.
  • MiRNA release kinetics are specific, with some reaching steady state within 24 hours, while others, like miR-16, did not equilibrate even after 3 days.

Conclusions:

  • Ex-miRNA stability is highly variable and significantly reduced under physiological conditions.
  • Intrinsic sequence properties largely determine ex-miRNA stability in biofluids.
  • Extracellular vesicle association confers significant stability to ex-miRNAs.
  • MiRNA-specific release and degradation kinetics have critical implications for their use as biomarkers and their role in gene regulation between cells.