microRNA分离制备方法的研究

兰州大学机构库 >生命科学学院

	microRNA分离制备方法的研究
Alternative Title	The Methods of MicroRNAs Isolation and Purification
	庞鑫
Thesis Advisor	廖世奇
	2012-05-22
Degree Grantor	兰州大学
Place of Conferral	兰州
Degree Name	硕士
Keyword	小鼠肝脏microRNA Poly (A)加尾法血清MicroRNA 克隆测序
Abstract	MicroRNA是一类18-24nt保守的非编码调控RNA家族，在真核细胞中通过与目的mRNAs的结合位点发生互补，进而在翻译水平上调控目的基因的表达。研究表明，microRNA在细胞增殖、分化、凋亡和肿瘤发生发展方面具有重要的调节功能。虽然针对microRNA及其功能的研究数目众多，但是提取分离microRNA的技术仍比较薄弱，尤其是在血清microRNA分离制备方面，需要进一步的探究。本文选取小鼠肝脏和人血清作为样本进行microRNA分离制备方法的研究。首先进行了小鼠肝脏microRNA的分离制备实验。使用RNAiso Plus获得小鼠肝脏总RNA后，采用Poly (A)加尾法进行实验：3'端加上多聚A尾，5'端连接一段L2连接子，并使用特异性引物Oligo(dT)，通过RT-PCR获得小分子RNA的cDNA文库并克隆测序。在成功提取小鼠肝脏microRNA的基础上，进行了人血清microRNA分离制备方法的研究。本实验使用RNAiso Plus提取获得人血清总RNA后，采用三种方法进行后续实验。本实验在小鼠肝脏中成功提取到4条microRNA序列，并将两条未知序列提交至GeneBank获得编号。在此基础上，对人血清microRNA提取分离方法进行了初步探索。实验中采用的Poly (A)法、连接子法和DraⅠ酶切法均存在一定的不足，需要进一步的改进。本实验为人血清microRNA分离制备方法的研究奠定基础。
Other Abstract	MicroRNAs are the conserved non-coding small RNAs family of 18-24 nucletides (18-24nt) in length. In eukaryotes, they regulate the expression of genes by complementary association with target mRNA at the translational level. The researches about microRNAs suggest that microRNAs are regulating functional on cell multiplication, differentiation apoptosis and the developing of tumor. Although there are many researches on microRNAs and their functions, the technoledge of microRNAs isolation and purification was ineffective, especially in serum microRNAs, which needs to be improved. In the study of microRNA isolation and purification, we choose mice liver tissue and serum as the experimental materials. Based on the mice liver experiment, we studied isolation and purification of human serum microRNAs. In summary, we obtained 4 microRNAs in mice liver, 2 of them unknown sequences were submitted into GeneBank. Based on it, we primarily explored the methods of human serum extraction and isolation. All of these 3 methods, poly (A) method, linker method and DraⅠmethod, have certain shortcomings and need to be improved. The study gave the basis to methods of extraction and isolation in human serum.
URL	查看原文
Language	中文
Document Type	学位论文
Identifier	https://ir.lzu.edu.cn/handle/262010/221944
Collection	生命科学学院
Recommended Citation GB/T 7714	庞鑫. microRNA分离制备方法的研究[D]. 兰州. 兰州大学,2012.

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