TY - JOUR T1 - Identification of reference genes for microRNAs of extracellular vesicles isolated from plasma samples of healthy dogs by ultracentrifugation, precipitation, and membrane affinity chromatography methods. AU - Narita,Momoko, AU - Nishida,Hidetaka, AU - Asahina,Ryota, AU - Nakata,Kohei, AU - Yano,Hirohito, AU - Ueda,Tomoyuki, AU - Inden,Masatoshi, AU - Akiyoshi,Hideo, AU - Maeda,Sadatoshi, AU - Kamishina,Hiroaki, PY - 2019/4/30/entrez PY - 2019/4/30/pubmed PY - 2019/6/21/medline SP - 449 EP - 454 JF - American journal of veterinary research JO - Am J Vet Res VL - 80 IS - 5 N2 - 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. SN - 1943-5681 UR - https://www.unboundmedicine.com/medline/citation/31034274/Identification_of_reference_genes_for_microRNAs_of_extracellular_vesicles_isolated_from_plasma_samples_of_healthy_dogs_by_ultracentrifugation_precipitation_and_membrane_affinity_chromatography_methods_ DB - PRIME DP - Unbound Medicine ER -