日本黑牛妊娠早期循环microRNA的分析

IF 1.9 2区 农林科学 Q2 AGRICULTURE, DAIRY & ANIMAL SCIENCE
K. Ono , S. Okamoto , C. Ninomiya , N. Toji , T. Kanazawa , T. Ishiguro-Oonuma , T. Takahashi , K. Iga , K. Kizaki
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引用次数: 0

摘要

循环microRNAs (miRNAs)已被用作人类和小鼠各种疾病和生理状况的生物标志物;对家畜,特别是牛的研究是有限的。在畜牧业中,早期妊娠诊断(特别是在母牛发情周期21天内)的重要性是非常高的。本研究比较了日本黑牛未妊娠和妊娠的循环miRNA,探讨了miRNA作为妊娠早期诊断的生物标志物,并建立了包括选择合适的参考miRNA和测定溶血对血浆miRNA定量影响的测量体系。在人工授精后第21天,从日本黑牛的血浆中提取miRNA,并使用定制的牛寡核苷酸芯片进行表达分析。选择差异表达的miRNA和参考候选miRNA,并使用逆转录定量PCR (RT-qPCR)进行验证。使用NormFinder软件选择合适的内源性参考miRNA进行归一化。为了评估溶血对miRNA定量的影响,采用4头处于发情周期的奶牛血浆制备溶血样品,并进行RT-qPCR。通过微阵列分析,在未怀孕和怀孕的奶牛中共检测到124种mirna。5种循环mirna的水平在怀孕奶牛中显著高于未怀孕奶牛,24种mirna仅在怀孕组中检测到。NormFinder分析和RT-qPCR验证显示,miR-2455是育成非妊娠和妊娠日本黑牛血浆中合适的参考miRNA,妊娠组miR-19b、miR-25、miR-29a和miR-148a均显著升高。这四种循环mirna在发情周期中没有变化,受溶血的影响较小。在目前的研究中,我们发现了四种mirna, miR-19b, miR-25, miR-29a和miR-148a,它们在怀孕的日本黑牛血浆中含量很高。由于这些mirna受溶血的影响较小,因此它们可能被用作牛早期妊娠诊断的生物标志物。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Analysis of circulating microRNA during early gestation in Japanese black cattle

Circulating microRNAs (miRNAs) have been used as biomarkers for various diseases and physiological conditions in humans and mice; studies in domestic animals, particularly cattle, are limited. The importance of early pregnancy diagnosis (especially within the 21-d cow estrous cycle) in the livestock industry is extremely high. This study compared the circulating miRNAs in bred non-pregnant and pregnant Japanese Black cows, explored miRNAs as biomarkers for early pregnancy diagnosis, and established a measurement system that included selecting an appropriate reference miRNA and determining the effect of hemolysis on miRNA quantification in plasma. miRNA was extracted from the plasma of Japanese Black cows on day 21 after artificial insemination and subjected to a customized bovine oligonucleotide microarray for expression analysis. Differentially expressed miRNAs and reference miRNA candidates were selected and validated using reverse transcription-quantitative PCR (RT-qPCR). An appropriate endogenous reference miRNA for normalization was selected using NormFinder software. To evaluate the effect of hemolysis on miRNA quantification, hemolyzed samples were prepared using plasma from four cows in the estrous cycle and subjected to RT-qPCR. A total of 124 miRNAs were detected in bovine plasma by microarray analysis in bred non-pregnant and pregnant cows. The levels of five circulating miRNAs were significantly higher in pregnant cows than in bred non-pregnant cows, and 24 miRNAs were detected only in the pregnant group. NormFinder analysis and RT-qPCR validation showed that miR-2455 was an appropriate reference miRNA in the plasma of bred non-pregnant and pregnant Japanese Black cows, and miR-19b, miR-25, miR-29a, and miR-148a were significantly higher in the pregnant group. These four circulating miRNAs did not change during the estrous cycle and were less affected by hemolysis. In the current study, we found four miRNAs, miR-19b, miR-25, miR-29a, and miR-148a, which were present at high levels in the plasma of pregnant Japanese Black cows. Since these miRNAs are less affected by hemolysis, they may potentially be used as biomarkers for early pregnancy diagnosis in cattle.

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来源期刊
Domestic animal endocrinology
Domestic animal endocrinology 农林科学-奶制品与动物科学
CiteScore
5.50
自引率
4.80%
发文量
58
审稿时长
31 days
期刊介绍: Domestic Animal Endocrinology publishes scientific papers dealing with the study of the endocrine physiology of domestic animal species. Those manuscripts utilizing other species as models for clinical or production problems associated with domestic animals are also welcome. Topics covered include: Classical and reproductive endocrinology- Clinical and applied endocrinology- Regulation of hormone secretion- Hormone action- Molecular biology- Cytokines- Growth factors
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