Comparison of RNA extraction and microRNA detection protocols for a small amount of germinal vesicle oocytes in bovine
Comparison of RNA extraction and microRNA detection protocols for a small amount of germinal vesicle oocytes in bovine
复制标题
牛少量生发囊泡卵母细胞RNA提取和microRNA检测方案的比较
DOI：
10.1016/j.anireprosci.2018.05.013
复制
发表时间：
2018-08-01
影响因子：
2.2
通讯作者：
Wang, Yongsheng
中科院分区：
文献类型：
--
作者：
Ma, Xiaonan;Zhang, Ying;Wang, Yongsheng
RT-qPCR是一种广泛使用的检测miRNA表达的方法。与mRNA相比，miRNA的长度较短，丰度较低，阻碍了可靠结果的获得。因此，miRNA的检测需要一种灵敏度高、准确度高的方法。对于家畜的卵母细胞和植入前的胚胎来说，收集大量的材料尤其困难。迫切需要建立一套适合检测微量此类物质的miRNA检测方法。本研究采用直接裂解法、miVana miRNA分离试剂盒和miRNasy Micro试剂盒提取了50个生发泡(GV)卵母细胞的总RNA。比较三组RNA的OD260/280值和浓度，以确定一种更好的RNA提取方法。此外，用实时定量聚合酶链式反应检测miRNA，比较了普通DNA和LNA引物的特异性和敏感性。结果显示，直接裂解组的RNA浓度显著高于其他两组。DNA和LNA的特异性相同，但LNA的敏感性高于DNA。这些结果表明，直接裂解结合LNA引物可能是一种适合于牛少量GV卵母细胞和植入前胚胎的miRNA检测的方法。
RT-qPCR is a widely used method to detect miRNA expression. Compared with mRNA, miRNA has a shorter length and lower abundance which hinders the acquisition of reliable results. Thus, miRNA detection requires a method with high sensitivity and accuracy. Collecting large amounts of material is particularly difficult for oocytes and pre-implantation embryos of domestic animals. Establishing a set of miRNA detection methods that are suitable to detect trace amounts of such materials is urgently needed. In this study, the total RNA in 50 germinal vesicle (GV) oocytes was isolated through direct lysis and by using mirVana miRNA Isolation Kit and miRNeasy Micro Kit. The OD260/280 values and concentrations of the RNA in these three groups were compared to identify a superior RNA isolation method. In addition, the specificity and sensitivity of common DNA and LNA primers were compared by real-time quantitative polymerase chain reaction for miRNA detection. Results show that the RNA concentration of in the direct lysis group was significantly higher than that in the other two groups. The specificity between the DNA primers and LNA primers was identical, whereas the sensitivity of LNA primers was superior to that of DNA primers. These results suggest that direct lysis combined with LNA primers might be a suitable protocol for the miRNA detection of a small amounts of GV oocytes and pre-implantation embryos in cattle.