改良Trizol法提取血浆游离RNA  

Extracting free RNA from plasma by modified Trizol methods

作　　者：陈雪梅[1] 李聪[1] 丁雨[1] 吴思思[1] CHEN Xue-mei;LI Cong;DING Yu;WU Si-si(Research Core Facility,West China Hospital,Sichuan University,Chengdu 610041,China)

机构地区：[1]四川大学华西医院公共实验技术中心,四川成都610041

出　　处：《生物技术》2020年第1期64-68,共5页Biotechnology

基　　金：国家自然科学基金资助项目(81870221)。

摘　　要：[目的]建立一种成本低、得率高的血浆游离RNA的提取方法。[方法]分别采用传统Trizol法、改良Trizol法、试剂盒法提取200μL健康成年人血浆中游离RNA,并比较三种方法所得RNA的浓度、纯度及大小片段RNA得率的差异。[结果]改良Trizol法所得RNA浓度是常规Trizol法的21.7倍(P<0.01),是试剂盒法的6.4倍(P<0.01);三种方法所得RNA纯度(A260/A280)无显著差异;采用RT-q PCR方法检测血浆游离RNA大小片段的回收率,外参秀丽线虫miR-39-3p(cel-miR-39-3p)代表小片段RNA,GAPDH代表大片段RNA,改良Trizol法检测Cq平均值分别为15.64和33.34,平均低于常规Trizol法2.05~3.62个Cq,低于试剂盒法0.32~3.34个Cq。[结论]改良Trizol法提取血浆游离RNA相较于常规Trizol法提高了10~20倍,增加了得率。[Objective]To establish a method for extracting plasma free RNA with low cost and high yield.[Method]The free RNA of 200μL healthy adult plasma were extracted by traditional Trizol methods,modified Trizol methods and kit methods,then comparing the concentration,purity and RNA extraction yield of RNA extracted by three methods.[Result]The RNA concentration obtained by the modified Trizol methods was 20.7 times(P<0.01)and 5.4 times(P<0.01)higher than the other two methods.To the purity of RNA(A260/A280),there were no significant difference in three methods.The RT-q PCR method was used to detect the recovery rate of large and small fragments.cel-miR-39-3 p represents small fragment RNAs,GAPDH represents large fragment RNAs.The average Cq value of modified Trizol methods were 15.64 and 33.34,which were 2.05-3.62 Cq lower than the conventional Trizol methods and 0.32-3.34 Cq lower than the Kit methods.[Conclusion]Compared with the conventional Trizol methods,the improved Trizol methods can increase the blood free RNA yield by 10-20 times.

关 键 词：TRIZOL法 RNA提取 血浆游离RNA 

分 类 号：Q781[生物学—分子生物学]