尼罗罗非鱼外周血白细胞总RNA的分离及反转录验证  

Total RNA isolation of Oreochromis niloticus peripheral leukocytes and quality verification by reverse transcription

作　　者：李超[1,2] 陈明[2,3] 黄维义[1] 王瑞[2] 余晓丽[2] 黄婷[2] 雷爱莹[2] 梁万文[2] 

机构地区：[1]广西大学动物科学技术学院,南宁530051 [2]广西水产研究所,南宁530021 [3]农业部水生动物遗传育种和养殖生物学重点开放实验室/中国水产科学研究院淡水渔业研究中心,江苏无锡214081

出　　处：《淡水渔业》2010年第2期19-23,共5页Freshwater Fisheries

基　　金：国家科技支撑计划课题(2008BADB9B04);农业部水生动物遗传育种和养殖生物学重点开放实验室开放基金(BZ2007-05);广西科技计划应用基础研究项目(桂科基0832029)

摘　　要：应用Trizol一步法对尼罗罗非鱼（Oreochromis niloticus）外周血白细胞总RNA提取方法进行优化,通过β-ac-tin基因反转录对所分离到的总RNA进行质量验证,并对mRNA的冻干浓缩进行了探讨,确定了一套快速、高效的尼罗罗非鱼外周血白细胞总RNA分离和mRNA浓缩体系。结果显示：获得的总RNA纯度较高,OD260/OD280均在1.9-2.0之间;甲醛变性电泳显示,总RNA完整性良好,28S与18S比值为2∶1;总RNA的分离量与起始白细胞量成正比,每毫升Trizol最大裂解量为1×10^8个白细胞,可得到总RNA量约为90μg;总RNA的分离量在前四个小时随着沉淀时间的增长而增加,并成功进行了β-actin基因的反转录。mRNA的分离率为1.08%,通过冻干浓缩成功使其浓度提高9倍。结果表明：分离到的总RNA质量可充分满足下游分子生物学实验要求,所建立的mRNA浓缩方法简单易行,可完全解决mRNA起始浓度低的难题。In this study,a fast,and efficient total RNA isolation and mRNA quality assay system from peripheral leukocytes of Oreochromis niloticus was established by optimization of one-step Trizol RNA extraction and quality verification by reverse transcription of the β-actin gene,and a method for RNA concentration with freeze-dry was also studied.The results showed that the total RNA isolated had a higher purity of OD260/OD280 at 1.9~2.0;Formaldehyde denaturing gel electrophoresis confirmed that the total RNA had good integrity,and the ratio of 28S∶18S at 2∶1;Isolated RNA quantity was proportional to the volume of the initial number of leukocytes and the largest cracker amount of per milliliter Trizol was 1×10^8,in which about 90ug total RNA could be yielded;Total RNA amount was increased proportional to the precipitation time in the first four hours;By using the isolated total RNAs,β-actin gene was successfully reverse transcribed.The mRNA isolation rate was 1.08% and its concentration was increased 9 times by freeze-dry.Therefore,the work revealed that the total RNA quality could meet the requirements of downstream molecular biology experiments,and the method for mRNA concentration was feasible and efficient.

关 键 词：尼罗罗非鱼(Oreochromis niloticus) 外周血白细胞 总RNA mRNA 冻干浓缩 

分 类 号：S965.125[农业科学—水产养殖] Q522[农业科学—水产科学]