激光微切割与定量PCR技术分析肾脏病理切片RNA  

Analysis of RNA In Pathological Sections of Kidney by Laser Capture Microdissection and Quantitative PCR

作　　者：吕杨[1] 尹忠[1] 王兆霞[1] 师锁柱[1] 洪权[1] 张雪光[1] 陈香美[1] 

机构地区：[1]中国人民解放军总医院肾病中心暨全军重点实验室,北京100853

出　　处：《中国生物化学与分子生物学报》2005年第5期695-699,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重大基础研究规化"973"项目资助(No.G2000057002);国家自然科学基金创新研究群体项目资助(No.30121005)~~

摘　　要：采用激光微切割与定量PCR技术,分析使用不同提取方法从不同固定方法固定的病理切片中提取的RNA.用70%乙醇、丙酮、甲醇、4%多聚甲醛固定肾脏冰冻切片,使用激光微切割技术切取肾小球,用硫氰酸胍方法(guanidinethiocyanatemethods,GTC)和Trizol试剂方法提取RNA,使用Taqman定量PCR方法分析比较各组RNA的量;选取丙酮固定的石蜡切片,使用激光微切割技术切取肾小球,采用RNA裂解液提取RNA,使用Taqman定量PCR方法,比较石蜡切片和冰冻切片中RNA含量.结果显示:提取沉淀性固定剂如乙醇、丙酮、甲醇固定的冰冻切片的RNA时,2种提取方法和3种固定方法对RNA含量的影响都无明显差异;但在提取4%多聚甲醛固定冰冻切片时,使用Trizol提取RNA含量明显高于使用GTC方法,且其含量与沉淀性固定剂固定的切片RNA含量无明显差异.石蜡切片中经激光微切割肾小球的RNA含量与冰冻切片经激光微切割肾小球的RNA含量无明显差异.结果提示:切片的固定方法和RNA的提取方法是影响切片RNA提取量的主要原因.The RNA in pathobiological sections fixed by several fixatives was analyzed by laser capture microdissection and quantitative PCR. slices. Frozen sections were fixed Kidneys were disposed to make frozen sections and paraffin-embedded sequentially by 70% ethanol, acetone, methanol, and 4% paraformaldehyde. The RNA in the 40 glomeruli captured by laser capture microdissection system （LCM） from frozen sections was isolated by two methods： guanidine thiocyanate methods （GTC） and Trizol agent methods. The RNA in glomeruli captured from acetone-fixed paraffin-embedded sections was isolated by RNA lysis buffer. The quality of GAPDH was measured by Taqman quantitative real-time PCR to reflect the quality of RNA. For RNA of glomeruli in frozen sections, no significant difference was found among sections which were fixed by precipitive fixatives such as methanol, ethanol, and acetone when using GTC or Trizol agent to extract RNA. However, for the frozen sections fixed by cross-linking fixative such as 4 % formaldehyde, Trizol agent was better than GTC to acquire more RNA which had no significant difference with precipitive fixatives fixed sections. When comparing RNA of glomeruli between acetone-fixed paraffin-embedded and frozen sections, there was no significant difference. It was concluded that the appropriate combination of RNA extract methods with fixatives plays the vital role in RNA acquisition.

关 键 词：激光微切割 定量PCR RNA 冰冻切片 石蜡切片 

分 类 号：Q7[生物学—分子生物学]