bta-miR-377琼脂糖凝胶电泳条件优化  

Optimization of bta-miR-377 Agarose Gel Electrophoresis Conditions

作　　者：郝琴琴 任静 成俊丽 李鹏飞[1] HAO Qinqin;REN Jing;CHENG Junli;LI Pengfei(College of Life Science,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学生命科学学院,山西太谷030801

出　　处：《山西农业科学》2022年第5期709-713,共5页Journal of Shanxi Agricultural Sciences

基　　金：国家自然科学基金项目(31873002);山西省应用基础研究计划面上项目(20210302123380);山西省现代农业产业技术体系建设专项。

摘　　要：bta-miR-377与下丘脑分泌的可卡因苯丙胺调节转录肽(Cocaine and amphetamine regulated transcript peptide,CART)存在靶向结合关系。为分析bta-miR-377在牛下丘脑组织中的表达情况,为后续探讨bta-miR-377与CART相互作用进而影响牛卵泡发育的作用机制奠定基础,经Trizol法提取牛下丘脑总RNA,采用茎环结构法设计特异性引物,并利用半定量RT-PCR技术克隆bta-miR-377,观察不同条件琼脂糖凝胶电泳后Marker和目的条带的分离情况,其中凝胶浓度为2.0%、2.5%;电压为100、80 V;时间为40、60、80 min;同时通过Image J软件对不同条件下获得的bta-miR-377条带进行灰度值分析,结合电泳结果确定最佳分离条件。结果显示,利用茎环结构法能够达到延长bta-miR-377片段的目的,且特异性强;凝胶浓度为2.5%、电压为80 V、时间为80 min时,bta-miR-377条带分离效果好且表达量高,该条件下条带的灰度值与其他组相比差异显著,与电泳分析结果一致,说明低浓度凝胶的分辨率低,长时间高压会导致DNA降解进而使条带弥散,而适当电压下电泳时间过短会使DNA分子无法聚集而使条带过宽,说明适宜的电泳条件是鉴定目的 miRNA表达的关键。综上可见,bta-miR-377在牛下丘脑中有表达,丰富了动物生殖生理学中下丘脑-垂体-性腺轴的内分泌调控理论。The targeted binding relationship between bta-miR-377 and hypothalamic secretion of cocaine and amphetamine regulated transcript peptide(CART) exists. To analyze the expression of bta-miR-377 in bovine hypothalamus for providing subsequent discussion of action mechanism of interaction between bta-miR-377 and CART, then influence on development of bovine ovarian follicle with foundation, in this study, total RNA from bovine hypothalamus was extracted by Trizol method,specific primers of bta-miR-377 were designed by stem-loop structure method, bta-miR-377 was cloned by semi quantitative RT-PCR technology, separation of Maker and target bands after different conditions of agarose gel electrophoresis were observed. The gel concentrations were 2.0%, 2.5%, voltage was 100, 80 V, time was 40, 60, 80 min. In addition, the gray ratio under different conditions of bta-miR-377 bands was analyzed by Image J software, the optimal separation condition was defined according to electrophoresis. The results showed that the stem-loop structure method could achieve the purpose of extending the fragment of bta-miR-377 with strong specificity. In particularly, the separation of bta-miR-377 was the optimal and expression was high when gel concentration was 2.5%, the voltage was 80 V, and time was 80 min. The gray value of the band in this condition was significantly different from other groups, which was consistent with the electrophoresis results,indicating that the resolution of low-concentration gel was low and long-term high-voltage would cause DNA degradation to make band dispersed.While the voltage was appropriate, even if the electrophoresis time was too short to aggregate the DNA molecules, resulting in the band wider. So the appropriate electrophoresis condition was the key to identify the expression of the target miRNA. In conclusion, bta-miR-377 was expressed in the bovine hypothalamus, and enriched the theory of endocrine regulation of the hypothalamus-pituitary-gonadal axis in animal reproductive physiology.

关 键 词：bta-miR-377 茎环结构法 半定量RT-PCR 琼脂糖凝胶 条件优化 

分 类 号：Q503[生物学—生物化学]