仿刺参体壁总RNA提取方法的建立

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。

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

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 one-step 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.