实时荧光定量PCR检测人肿瘤细胞NKG2D配体基因表达  

Real-Time Fluorescence Quantitative PCR Assay for Human NKG2D Ligand mRNA Quantification

作　　者：刘枫[1] 郑冰蓉[1] 杨举伦[2] 王力[2] 陈玥[2] 赵稳兴[2] 

机构地区：[1]云南大学生命科学学院,云南昆明650091 [2]解放军昆明总医院病理科,云南昆明650032

出　　处：《现代生物医学进展》2011年第19期3621-3624,共4页Progress in Modern Biomedicine

基　　金：云南省自然科学基金(2010ZC186)

摘　　要：目的:建立人肿瘤细胞NKG2D配体基因(MICA、MICB、ULBP1、ULBP2、ULBP3)表达的实时荧光定量PCR(real-timefluorescence quantitative PCR)检测方法。方法:根据NCBI基因库中NKG2D配体基因序列,设计合成引物。用Trizol法从培养的肿瘤细胞(BEC-7402、HeLa、MDA-MB-435、XWLC-05)中提取总RNA,逆转录成cDNA,建立实时荧光定量PCR检测NKG2D配体基因表达的方法,并检测NKG2D配体在肿瘤细胞株中的表达。结果:经过琼脂糖凝胶电泳、熔解曲线和标准曲线分析,用所设计的引物和SYBR Green I能够特异扩增和定量检测NKG2D配体基因的表达。该方法成功检测4种肿瘤细胞NKG2D配体基因的表达。结论:建立了人NKG2D配体基因表达的实时荧光定量PCR检测方法,为进一步研究人NKG2D配体在肿瘤免疫中的作用提供了有效手段。Objective： This study aimed to develop a real-time fluorescence quantitative PCR assay for NKG2D ligand mRNA in human. Methods： The primers for NKG2D ligands and keeping-home gene GAPDH were designed and synthesized according to the gene sequences from NCBI, The total RNA of tumor cells was extracted by Trizol protocol, and eDNA was synthesized by reverse transcriptase, then the real-time fluorescence quantitative PCR assay was established using SYBR Green I. Results： The method was efficient and sensitive for NKG2D ligand detection, which was assessed using standard curve, melting curve and gel electrophoresis analysig Conelusion： We successfully detected the NKG2D ligand mRNA in tumer cell lines such as BEC-7402, HeLa, MDA-MB-435 and XWLC-05 by the method.

关 键 词：NKG2D配体 实时荧光定量PCR 肿瘤细胞 

分 类 号：R392.11[医药卫生—免疫学] R730.231[医药卫生—基础医学]