实时RT-PCR基因表达相对定量REST~软件分析与2^((-ΔΔCT))法比较  

Quantitative Analysis of Real-Time PCR Expression Production by REST and 2^((-ΔΔCT))

作　　者：庾蕾[1] 刘建平[1,2] 庄志雄[1] 杨淋清[1] 张仁利[1] 叶小明[1] 程锦泉[1] 

机构地区：[1]深圳市疾病预防控制中心分子生物学研究室 [2]华中科技大学同济医学院公共卫生学院,武汉430030

出　　处：《热带医学杂志》2007年第10期956-958,共3页Journal of Tropical Medicine

基　　金：深圳市科技计划项目(No.TH200505300499A)。

摘　　要：目的利用实时RT-PCR技术建立一种简便、准确的基因表达相对定量方法。方法以正常的人支气管上皮细胞株(16HBE)cDNA为模板5倍系列稀释,分别作DNA聚合酶K(POLK)和磷酸甘油醛脱氢酶(GAPDH)基因标准曲线。以不同剂量反式二羟环氧笨并芘(anti-BPDE)诱导的16HBE、anti-BPDE转化的16HBE及肺癌细胞株(H1299)cDNA为模板,进行实时定量RT-PCR。以GAPDH基因作为内参照,进行POLK基因表达相对定量。实验数据分别采用2(-ΔΔCT)法及REST软件分析。结果5倍系列稀释法制作的标准曲线,呈现较好的线型关系(Rsq0.997)。POLK及GAPDH基因扩增效率分别为117.5%和103.5%。采用2(-ΔΔCT)法计算出的表达比值均比REST软件分析高。结论实时RT-PCR技术结合REST软件分析是一种简便、准确的基因表达相对定量分析手段。Objective To develop a simple and accurate method for the quantification of real-time RT-PCR gene products. Method Standard curves of POLK and GA PDH genes were made from 5-fold serially diluted eDNA samples from the normal 16HBE cells. The levels of POLK mRNA in the malignant transformed 16HBE cells and human lung cancer cells （H1299） treated with various concentrations of BPDE were determined. GAPDH gene was used as the reference gene. The relative expression levels were analyzed by REST and 2^（-△△CT） method. Result A good linear relationship（Rsq 0.997） was obtained from the standard curves. The efficiency of amplification of POLK and GAPDH genes was 117.5% and 103.5%, respectively. The expression ratios calculated by using 2^（-△△CT） method were higher than the rations obtained from the REST. Conclusion Real-time RT-PCR in combination with REST is a simple and accurate method for relative quantification of gene expression.

关 键 词：实时RT—PCR 相对定量 标准曲线 基因表达 

分 类 号：R446.61[医药卫生—诊断学]