激光显微切割分离细胞的微量RNA质量鉴定体系的建立  

Establishment of the pipeline for RNA quality assessment from the cells obtained by laser capture microdissection

作　　者：杨燕青[1,2] 张雯[2] 张宝峰[2] 郜恒骏[2] 张庆华[1,2] 

机构地区：[1]上海交通大学医学院附属瑞金医院上海血液学研究所,医学基因组学国家重点实验室,上海200025 [2]生物芯片上海国家工程研究中心,上海201203

出　　处：《遗传》2008年第11期1521-1526,共6页Hereditas（Beijing)

基　　金：国家重大科学研究计划项目(973计划)(编号:2006CB910402);国家高科技发展计划项目(863计划)(编号:2002AA2Z2002);上海市科技发展基金计划项目(编号:05XDB1404;0402H1P19;07JC14041);浦东新区科技发展基金项目(编号:PKJ-2005-49)资助~~

摘　　要：探索一套激光显微切割(Laser capture microdissection,LCM)分离细胞后获得的微量RNA质量鉴定标准操作流程。选取3个低温保存的胃癌旁组织样本,冰冻切片进行甲酚紫染色和病理学检查,利用激光显微切割技术分离非癌上皮细胞,提取RNA并以Agilent 2100生物分析仪鉴定RNA的纯度和完整性。同时,选择高、中、低3种不同表达丰度的6个基因(EF1A,ACTB,GAPHD,B2M,MED1,CK20),在每个基因的5′和3′端设计引物,RT-PCR扩增。以3个培养细胞制备的高质量RNA和3个有降解的胃癌旁组织样本RNA作对照,RT-PCR扩增结果与Agilent 2100生物分析仪的结果高度一致。结果显示冻存组织进行冰冻切片结合病理学检查后,LCM获取细胞提取微量RNA采用RT-PCR进行质量鉴定是一种操作简单的稳定方法,可以作为肿瘤基因组研究的有效和常规方法。We developed a standard protocol for quality assessment of low amount RNA from the cells obtained by laser capture microdissection （LCM）. Three gastric noncancerous tissues were cryo-sectioned, stained with Cresyl Violet, and pathologically rechecked. Epithelial cells were obtained by LCM and RNA was isolated. Agilent 2100 bioanalyzer was used to check the RNA quality. To validate the results from 2100 bioanalyzer, RT-PCR was performed with six genes at both 5＇ and 3＇ end-regions of different abundance （EFIA and ATCB of high abundance, GAPDH and B2M of moderate abundance, and MED1 and CK20 of low abundance）. RT-PCR analysis of 3 good quality RNAs from cultured cell lines and 3 poor quality RNAs from gastric noncancerous tissues showed high correlations with that from 2100 bioanalyzer. In conclusion, the pipeline for low amount RNA quality assessment by RT-PCR from tissue cryo-section, pathological recheck, LCM purification and RNA isolation is applicable as a routine method in cancer genome research.

关 键 词：激光显微切割 RT-PCR RNA完整性 质量鉴定 

分 类 号：Q343[生物学—遗传学]