Identification of reference genes for microRNAs of extracellular vesicles isolated from plasma samples of healthy dogs by ultracentrifugation, precipitation, and membrane affinity chromatography methods
Identification of reference genes for microRNAs of extracellular vesicles isolated from plasma samples of healthy dogs by ultracentrifugation, precipitation, and membrane affinity chromatography methods
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DOI：
10.2460/ajvr.80.5.449
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发表时间：
2019-05-01
影响因子：
1
通讯作者：
Kamishina, Hiroaki
中科院分区：
文献类型：
--
作者：
Narita, Momoko;Nishida, Hidetaka;Kamishina, Hiroaki
目的比较用于从犬血浆样品中分离细胞外囊泡（EV）的超离心、沉淀和膜亲和色谱方法，并鉴定用于掺入健康狗血浆EV中microRNA表达的定量逆转录PCR测定的合适的参考基因。通过超离心、沉淀和膜亲和色谱法从0.3mL这些样品中分离EV。进行纳米颗粒跟踪分析以确定通过超离心方法分离的EV的浓度和尺寸分布。然后通过定量逆转录PCR测定比较4种microRNA（let-7a、miR-16、miR-26 a和miR-103）的表达水平（循环阈值）。三种统计程序用于鉴定最适合用作参考基因的microRNA。结果表明，超离心法是从0.3 mL血浆中分离microRNA的所有三种方法中最稳定的。纳米颗粒跟踪显示，通过超离心法获得的EV样品含有约1.59 X 10（10）个囊泡/mL +/- 4.2 X 10（8）个囊泡/mL的平均值+/- SD。结论和临床相关性超离心法有潜力作为一种稳定的方法，用于从犬血浆样品中分离EV，具有高回收率，和miR-103可能提供最稳定的参考基因，用于归一化与超离心分离的血浆EV相关的microRNA表达数据。
OBJECTIVETo 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.ANIMALS6 healthy Beagles.PROCEDURESPlasma 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.RESULTSResults 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 X 10(10) vesicles/mL +/- 4.2 X 10(8) vesicles/mL. Of the 4 microRNAs in plasma EVs isolated by ultracentrifugation, miR-103 was the most stable.CONCLUSIONS AND CLINICAL RELEVANCEThe 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.