单层培养细胞总RNA两种提取方法的比较  

Comparison of the cell collection by two methods for total RNA extraction from monolayer cultured cells

作　　者：于伟[1] 董霁[2] 王丽峰[2] 赵黎[2] 高亚兵[2] 彭瑞云[2] 

机构地区：[1]解放军总医院心血管外科,北京100853 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《中国体视学与图像分析》2014年第4期387-391,共5页Chinese Journal of Stereology and Image Analysis

基　　金：国家自然科学基金资助(81172620)

摘　　要：目的探讨单层培养细胞总RNA提取过程中先行细胞消化或离心富集对所提RNA质量是否产生影响。方法培养NGF诱导的PC12细胞,采用Trizol试剂提取RNA:离心组(A)先将贴壁生长细胞经胰酶消化、离心获得细胞沉淀,加入1 ml Trizol进行RNA提取;直接组(B)将培养液弃尽后直接加入Trizol试剂(1 ml/10 cm2瓶壁)进行RNA提取;均采用琼脂糖凝胶电泳分析RNA完整性,紫外分光光度仪测定RNA浓度和OD260/OD280比值。结果凝胶电泳结果显示,直接组出现3条清晰的条带(28S、18S和5S条带),离心组在电泳末端5S区出现粗大的条带。两组OD260/OD28比值相似,约为1.6。直接组组间样品间RNA浓度相差较大。结论采用离心法和直接法提取贴壁细胞总RNA,先行细胞离心富集增加了RNA降解机会,直接法提取的RNA质量较高,考虑对后续实验的影响,直接法更优于离心法。Objective The purpose of this study was to investigate the effect of cell collection by direct dissociation or centrifugation on the total RNA extraction from monolayer cultured cells. Methods PC12 cells were cultured after NGF induction. Trizol was used as the RNA extraction reagent. The method used in the group A was to collect cells with trypsin dissociation and centrifugation before adding lml Trizol. The method used in the group B was to add Trizol into culture flask directly （ 1 ml/10 cm2）. The integri- concentration and purity of isolated RNA were analyzed. Results The results of electrophoresis showed that the group B had three clear bands （28S, 18S and 5S） and the group A had one wide band at 5S. It indicates that the RNA extracted from the group B was integral but RNA from the group A had been degraded. The ratios of OD260/OD2s0 of both groups were about 1.6. The RNA concentration of B1 was lower than other samples. Conclusions Not to collect cells before adding the extraction reagent is an op- timal method to extract the total RNA from monolayer cultured cells, which can better ensure the integrity of isolated RNA. In order to keep the quantity of RNA, the extraction reagent should be reacted with cultured cells sufficiently

关 键 词：单层培养细胞 RNA提取 离心法 直接法 

分 类 号：R346[医药卫生—基础医学]