旋毛虫肌幼虫总RNA提取方法研究初报

【研究目的】探索高质量的旋毛虫(Trichinella spiralis)肌幼虫总RNA提取方法；【方法】利用Trizol试剂采用三种方法提取旋毛虫肌幼虫总RNA。(1)液氮预冷的研钵中研磨新鲜虫体至样品快融化时，加入适量的Trizol，继续研磨至液状后转移到离心管中；(2)A将新分离的虫体放到-20℃预冷的研钵中，在研磨过程中不断地添加液氮以保持虫体冷冻，之后对冷冻状的样品粉末继续操作；B把液氮冻存的新鲜旋毛虫肌幼虫取出，放到-20℃预冷的研钵中重复A的操作；(3)新鲜虫体放入匀浆器中，加适量Trizol，在冰水混合物中研磨20分钟。虫体破碎后按Trizol说明书继续抽提。最后通过凝胶电泳和紫外吸收光度值检测。【结果】用液氮和Trizol结合的方法，使用新鲜样品提取旋毛虫肌幼虫总RNA，在纯度和得率上都较为理想；【结论】使用新鲜的样品，且液氮与裂解液结合，是获得高质量RNA的可行方法。

Study in Extracting Method of Total RNA of Muscle Larvae of Trichinella spralis

【OBJECTIVE】In order to explore the method of extracting high quality total RNA from muscle larvae of T.spiralis【METHOD】Total RNA of muscle larvae of T.spiralis were extracted by three methods of using Trizol agentv, (After washing worms with DEPC-treated water, spin down worms at 10000g in microfuge for 1 minute and then remove excess water. Total RNA of muscle larvae of T.spiralis were extracted by three methods of using Trizol reagent:(1)method combining liquid nitrogen and Trizol : After putting liquid nitrogen into mortar up to T.spiralis prepared can be frozen ,put fresh samples into mortar and quickly grinding and add 1ml of Trizol Reagent per 50~100 mg of T.spiralis before thawing ,and transfer the liquid into the eppendorf tubev(2)liquid method: A: Put the fresh samples into mortar precooled at -20℃, In the course of of grinding, add liquid nitrogen unceasing to keep samples frozen and then transfer the powders into the eppendorf tube prepared. B: Put fresh T.spiralis into liquid nitrogen and store at -80℃ prior to grinding, the rest procedure is the same as A.(3)homogenization method: Homogenize T.spiralis samples in 1 ml of Trizol reagent per 50~100 mg of tissue for 20 minutes using a glass-homogenizer putting ice water mixture. The protocols are carried according to the methods of Trizol after grinding or homogenating. The total RNA were compared by gel electrophoresis and the ratio of OD260/OD280)【RESULT】The method of combining liquid nitrogen and Trizol was the suitable method in extracting total RNA from muscle larvae of T.spiralis and obtained the best purification and yield of total RNA; 【CONCLUSION】The high quailty total RNA can be obtained with fresh (samples) and combining liquid nitrogen and lysate.