文章摘要
刘正霞,徐 阳,徐进梅,杨明夏,马 磊,朱昌亮.不同引物及数据分析方法对定量PCR结果的影响[J].南京医科大学学报,2009,29(8):1112~1117
不同引物及数据分析方法对定量PCR结果的影响
The Effects of different primers and data analysis methods on real-time PCR
投稿时间:2009-02-06  
DOI:10.7655
中文关键词: 实时荧光定量PCR  2-△△CT法  Pfaffl法  相对标准曲线法  扩增效率
英文关键词: quantitative real-time PCR  2(-DeltaDeltaC(T)) method  Pfaffl method  relative standand curve method  amplification efficiency
基金项目:国家自然科学基金项目(30671827和30628022)和科技部自然资源平台项目(2005DKA21104)
作者单位
刘正霞 南京医科大学病原生物学系,江苏省现代病原生物学重点实验室,江苏 南京 210029 
徐 阳  
徐进梅  
杨明夏  
马 磊  
朱昌亮  
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中文摘要:
      目的:研究不同引物及数据分析方法对实时荧光定量PCR SYBR Green染料法检测基因表达差异结果分析的影响?方法:通过Primer Express 2.0软件对目的基因CCND1?C-JUN分别设计4对不同引物进行SYBR Green染料法定量PCR扩增,并分别使用2-△△CT法?Pfaffl法及相对标准曲线法计算肺癌紫杉醇敏感细胞株及耐药细胞株间目的基因的表达差异,分析不同引物和数据计算方法对实验结果的影响?结果:统计学分析表明,引物的扩增效率对定量PCR结果分析有显著影响,不同引物特异性扩增得到的样本间表达倍数有显著差异(P < 0.05)?3种计算方法中,Pfaffl法与相对标准曲线法无统计学差异(P > 0.05),而2-△△CT法与其他2种方法均有显著差异(P < 0.05)?结论:引物的选择对于定量PCR结果有显著影响;Pfaffl法是更准确,更合理的相对定量计算方法?
英文摘要:
      Objective:To study the effects of different primers and data analysis methods on quantitative real-time PCR. Methods: Four pairs of different primers were designed for target genes CCND1 and C-JUN using Primer Express 2.0 software. These primers were then used for quantitative PCR amplification and the differences in expression levels between paclitaxel sensitive and resistant lung cancer cell lines were calculated using 2(-DeltaDeltaC(T))method,Pfaffl method and the relative standard curve method,respectively. Results:The expression levels obtained from different primers were significantly different (P < 0.05). In the three kinds of calculation methods,Pfaffl method has no significant difference with the relative standard curve method (P > 0.05),however,2(-DeltaDeltaC(T))method has significant differences with other two kinds of methods (P < 0.05). Conclusion:The amplification efficiency of primers have significant impact on quantitative PCR analysis. The Pfaffl analysis method in the evaluation of relative real-time PCR may be more accurate.
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