坛紫菜叶状体RNA提取方法的改良与比较  

Improvement and comparison of extraction methods of total RNA from thalli in Porphyra haitanensis

作　　者：谢潮添[1] 梅高尚[1] 陈昌生[1] 纪德华[1] 

机构地区：[1]集美大学水产学院,福建厦门361021

出　　处：《水产学报》2010年第2期220-226,共7页Journal of Fisheries of China

基　　金：国家自然科学基金项目(40676077;40806065);国家"八六三"高技术研究发展计划(2006AA10A413);福建省科技平台建设项目(2007N2011);福建省自然科学基金项目(2007J0064)

摘　　要：为提取高质量的坛紫菜叶状体总RNA,对常用的几种植物RNA提取方法(CTAB法、SDS法、异硫氰酸胍法)按照去除多糖、多酚的方法进行了改良,并对分离的总RNA根据吸光值、电泳图谱及RT-PCR检测等结果与两种试剂盒(离心柱试剂盒法,RNAiso法)的提取结果进行了比较。结果表明,SDS法、异硫氰酸胍法和RNAiso法3种方法提取的RNA纯度低,质量差,部分RNA已经发生了降解。而改良CTAB法和离心柱试剂盒法提取的总RNA质量可靠,完整性好,纯度高,并且成功去除了可能影响逆转录酶活性的物质,可以进一步应用于cDNA文库构建,基因表达分析等后续实验,但这两种方法各有优缺点,应根据实际情况选用合适方法进行坛紫菜叶状体总RNA的分离。In order to develop a simple and efficient protocol for isolating total RNA from thalli of Porphyra haitanensis, three methods （CTAB method, SDS method, Guanidine thiocyante method） which were commonly used to isolate and purify total RNA were improved based on the methods of wiping off polysaccharides and polyphenol, and absorbency method, electrophoresis method and RT-PCR method were employed to determine the quality and quantity of RNA. RNA isolated by spin column kit method and RNAiso method was also compared with the RNA by the three methods. The results show the total RNA isolated by SDS method, guanidine thiocyante method and RNAiso method was of poor purity, retained some protein and polysaccharide and some RNA have dissolved into single nucleotide acid. But the total RNA which isolated by CTAB method and spin column kit method was of good quality and high purity, the substance which can restrain the active of reverse transcription enzyme also was cleared. These results suggest that CTAB method and spin column kit method are fit for the isolation of total RNA from the thalli of P. haitanensis, but each of the two methods has its advantages and disadvantages, we should choose the appropriate method based on the situation.

关 键 词：坛紫菜 叶状体 RNA提取 RT—PCR 

分 类 号：Q946[生物学—植物学] S917[农业科学—水产科学]