激光显微切割分离细胞的微量RNA质量鉴定体系的建立-遗传.PDFVIP

HEREDITAS (Beijing) 2008 11 , 30(11): 1521―1526 ISSN 0253-9715212 技术与方法 DOI: 10.3724/SP.J.1005.2008.01521 RNA 1, 2 2 2 2 1, 2 杨燕青 , 张雯 , 张宝峰 , 郜恒骏 , 张庆华 1. , , 200025; 2. , 201203 : 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。 : 激光显微切割; RT-PCR; RNA 完整性; 质量鉴定 Establishment of the pipeline for RNA quality assessment from the cells obtained by laser capture microdissection 1, 2 2 2 2 YANG Yan-Qing , ZHANG Wen , ZHANG Bao-Feng , GAO Heng-Jun , ZHANG Qing-Hua1, 2 1. State Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 2. National Engineering Center for Biochip at Shanghai, Shanghai 201203, China Abstract: 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 bioan