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  • Author or Editor: Hirohito Yano x
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Abstract

OBJECTIVE

To compare ultracentrifugation, precipitation, and membrane affinity chromatography methods for isolation of extracellular vesicles (EVs) from canine plasma samples and to identify suitable reference genes for incorporation into a quantitative reverse transcription PCR assay of microRNA expression in plasma EVs of healthy dogs.

ANIMALS

6 healthy Beagles.

PROCEDURES

Plasma samples were obtained from each dog, and EVs were isolated from 0.3 mL of these samples via ultracentrifugation, precipitation, and membrane-affinity chromatographic methods. Nanoparticle tracking analysis was performed to determine the concentration and size distribution of EVs isolated by the ultracentrifugation method. Expression levels (cycle threshold values) of 4 microRNAs (let-7a, miR-16, miR-26a, and miR-103) were then compared by means of quantitative reverse transcription PCR assay. Three statistical programs were used to identify the microRNAs most suitable for use as reference genes.

RESULTS

Results indicated that ultracentrifugation was the most stable of all 3 methods for isolating microRNAs from 0.3 mL of plasma. Nanoparticle tracking revealed that EV samples obtained by the ultracentrifugation method contained a mean ± SD of approximately 1.59 × 1010 vesicles/mL ± 4.2 × 108 vesicles/mL. Of the 4 microRNAs in plasma EVs isolated by ultracentrifugation, miR-103 was the most stable.

CONCLUSIONS AND CLINICAL RELEVANCE

The ultracentrifugation method has potential as a stable method for isolating EVs from canine plasma samples with a high recovery rate, and miR-103 may provide the most stable reference gene for normalizing microRNA expression data pertaining to plasma EVs isolated by ultracentrifugation.

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in American Journal of Veterinary Research

Abstract

OBJECTIVE

To measure expression of microRNAs (miRNAs) in plasma and in extracellular vesicles (EVs) derived from plasma for dogs with glioma and dogs with other brain diseases.

SAMPLE

Plasma samples from 11 dogs with glioma and 19 control dogs with various other brain diseases.

PROCEDURES

EVs were isolated from plasma samples by means of ultracentrifugation. Expression of 4 candidate reference miRNAs (let-7a, miR-16, miR-26a, and miR-103) and 4 candidate target miRNAs (miR-15b, miR-21, miR-155, and miR-342-3p) was quantified with reverse transcription PCR assays. Three software programs were used to select the most suitable reference miRNAs from among the 4 candidate reference miRNAs. Expression of the 4 target miRNAs was then calculated relative to expression of the reference genes in plasma and EVs, and relative expression was compared between dogs with glioma and control dogs with other brain diseases.

RESULTS

The most suitable reference miRNAs were miR-16 for plasma and let-7a for EVs. Relative expression of miR-15b in plasma and in EVs was significantly higher in dogs with glioma than in control dogs. Relative expression of miR-342-3p in EVs was significantly higher in dogs with glioma than in control dogs.

CONCLUSIONS AND CLINICAL RELEVANCE

Results suggested that miR-15b and miR-342-3p have potential as noninvasive biomarkers for differentiating glioma from other intracranial diseases in dogs. However, more extensive analysis of expression in specific glioma subtypes and grades, compared with expression in more defined control populations, will be necessary to assess their clinical relevance.

Restricted access
in American Journal of Veterinary Research