摘要
目的探讨适合现代医学实验室的高效组织总RNA提取方法,以及组织样本储存时间对后续实时荧光定量聚合酶链反应(qRT-PCR)检测有效性的影响。方法分别采用全自动样本处理器+磁珠法全自动核酸提取(方法1)、全自动样本处理器+离心柱法(方法2)、手工匀浆器+磁珠法全自动核酸提取(方法3)和手工匀浆器+离心柱法(方法4)4种方法制备小鼠新鲜心、肝、肾组织总RNA,比较各方法制备的总RNA质量。评价方法1和方法4制备的新鲜、-80℃储存1年和2年的小鼠肝组织总RNA质量及其对qRT-PCR检测结果的影响。结果方法1制备的小鼠新鲜组织总RNA浓度、完整性均优于其他3种方法。方法1制备的1年和2年肝组织总RNA完整性均明显优于方法4。3个不同储存时间肝组织样本的Ct值范围分别为18~19、21~22和27~28,2年肝组织样本总RNA降解明显。结论方法1是最佳的组织总RNA制备方法,尤其针对保存2年内的稀有生物样本。用于qRT-PCR检测的生物样本于-80℃储存时间最好不超过1年。
Objective Toexplore the efficient methods of tissue total RNA extraction suitable for modern medical laboratories,and the effect of tissue sample storage time on the effectiveness of subsequent qRT-PCR detection.Methods The total RNA of mouse fresh heart,liver and kidney tissues were prepared by four methods,namely,automatic sample processor combined with magnetic beads method(method 1),automatic sample processor combined with centrifugal columns method(method 2),manual homogenizer combined with magnetic beads method(method 3)and manual homogenizer combined with centrifugal columns method(method 4).The quality of total RNA prepared by various methods wascompared.Finally,the total RNA quality of fresh,one and two years storage at-80℃in mouse liver tissue prepared by methods 1 and 4 and its influence on qRT-PCR detection results were evaluated.Results The total RNA concentration and integrity of mouse fresh tissue prepared by method 1 were better than those of the other three methods.The integrity of total RNA prepared by method 1 for one year and two years of liver tissue was significantly better than that of method 4.The Ct values of liver tissue samples at three different storage times were 18-19,21-22 and 27-28,respectively.The total RNA of two years liver tissue sample was significantly degraded.Conclusion Method 1 is the best preparation method of tissue total RNA,especially for rare biological samples stored for two years.The biological samples used for qRT-PCR detection should be stored at-80℃for no more than one year.
作者
刘江丽
易旭
吴雪莉
刘蕊
王硕石
陈旭
LIU Jiangli;YI Xu;WU Xueli;LIU Rui;WANG Shuoshi;CHEN Xu(Clinical Medicine Laboratory,the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang,Guizhou 550003,China;the Second Clinical Medical College,Guizhou University of Traditional Chinese Medicine,Guiyang,Guizhou 550025,China)
出处
《重庆医学》
CAS
2023年第11期1614-1619,共6页
Chongqing medicine
基金
国家自然科学基金项目(81660752)
贵州中医药大学第二附属医院青苗科研启动基金项目(GZEYK-Y[2022]14号)。
