仿刺参体壁总RNA提取方法的建立  

A Method Established for Total RNA Isolation from the Body Wall of Apostichopus japonicus

作　　者：郑珂[1] 陈秋实[1] 李霞[1] 

机构地区：[1]大连水产学院农业部海水增养殖学与生物技术重点开放实验室,辽宁大连116023

出　　处：《生物技术通讯》2007年第1期84-87,共4页Letters in Biotechnology

基　　金：国家自然科学基金项目(30371099);辽宁省自然科学基金项目(20052139)

摘　　要：目的:通过对TRIzol一步法进行改进,建立一种从富含胶原蛋白、多糖及色素的仿刺参体壁提取总RNA的有效方法。方法:样品在液氮中研磨并用TRIzol匀浆后再进行抽提;对TRIzol一步法提取的总RNA进行DNaseⅠ消化和酚氯仿抽提,用2.5mol/L的醋酸钾沉淀,并加入适量糖原(10mg/mL)与RNA共沉淀。结果:琼脂糖凝胶电泳和紫外分光光度法以及RT-PCR检测结果表明,改进的方法能够有效去除基因组DNA、蛋白、多糖及色素的污染,RNA的产率提高。结论:制备的总RNA纯度高,完整性好,能够满足mRNA差异显示RT-PCR等分子生物学研究的要求,是一种提取仿刺参体壁及其他富含黏多糖、胶原蛋白和色素的动物组织总RNA的有效方法。Objective： To establish a method for total RNA isolation from Apostichopus japonicus＇ body wall rich in collagen, polysaccharides and pigments by improving TRIzol one-step method. Methods： Materials were triturated in liquid nitrogen immediately followed by homogenization in TRIzol regent before extraction. Total RNA isolated with TRIzol onestep method was treated with an additional DNase digestion, hydroxybenzene（saturated with water）-chloroform（5：1） extraction, 2.5 mol/L potassium acetate precipitation and glycogen（10 mg/mL） co-precipitation. Results： Being detected by agarose gel electrophoresis, UV spectrophotometer and RT-PCR, total RNA isolated with the improved method was proved to be free of the contamination of genomic DNA, protein, polysaccharides and pigments. The yields of total RNA were increased. Conclusion： Total RNA was pure and intact enough for molecular research such as mRNA differential display RT-PCR. It is an effective method for total RNA isolation from animal tissues rich in polysaccharides, pigments and collagen.

关 键 词：仿刺参 体壁 总RNA提取 

分 类 号：Q503[生物学—生物化学] Q781