| 刘枫1 郑冰蓉1 杨举伦2 王力2 陈玥2 赵稳兴2.实时荧光定量PCR 检测人肿瘤细胞NKG2D 配体基因表达[J].,2011,11(19):3621-3624 |
| 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 LigandmRNA Quantification |
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| DOI： |
| 中文关键词: NKG2D 配体 实时荧光定量PCR 肿瘤细胞 |
| 英文关键词: NKG2D ligand Real-time fluorescence quantitative PCR Tumor cell |
| 基金项目:云南省自然科学基金（2010ZC186） |
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| 中文摘要: |
| 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-time
fluorescence 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。 |
| 英文摘要: |
| 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 cDNA 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 analysis. Conclusion:
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. |
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