Taqman荧光定量PCR是核酸水平对管家基因GAPDH进行定量的好方法

首都医科大学学报 ›› 2006, Vol. 27 ›› Issue (2): 210-213.

Taqman荧光定量PCR是核酸水平对管家基因GAPDH进行定量的好方法

王萍, 丛敏, 李忆梅, 唐淑珍, 刘晓明, 王宝恩, 贾继东, 尤红

首都医科大学附属北京友谊医院肝病研究中心

收稿日期:2005-06-20 修回日期:1900-01-01 出版日期:2006-04-24 发布日期:2006-04-24
通讯作者: 尤红

Taqman Fluorescence Quantitative Polymerase Chain Reaction: A Good Method for Housekeeping Gene GAPDH Quantification

Wang Ping, Cong Min, Li Yimei, Tang Shuzhen, Liu Xiaoming, Wang Baoen, Jia Jidong, You Hong

Liver Research Center, Beijing Friendship Hospital, Capital University of Medical Sciences

Received:2005-06-20 Revised:1900-01-01 Online:2006-04-24 Published:2006-04-24

摘要/Abstract

摘要： 目的比较Taqman荧光定量PCR法、SYBR Green I荧光定量PCR法和普通PCR法制作3-磷酸甘油醛(GAPDH)标准曲线的检出下限、线性范围的差异,确定一种较好的GAPDH绝对定量方法.方法构建含GAPDH全长的质粒,经PCR和EcoR I限制性酶切鉴定确认后,紫外定量并连续稀释10倍作为标准品.分别用Taqman法和SYBR Green I法于荧光定量PCR仪上制作标准曲线,同时用普通PCR结合琼脂糖凝胶电泳利用Quantity One软件定量并制作标准曲线.结果 Taqman法检出GAPDH的下限为2.0×10⁴拷贝,线性范围:2.0×10⁴～2.0×10¹⁰拷贝;SYBR Green I法检出GAPDH的下限为2.0×10⁷拷贝,线性范围:2.0×10⁷～2.0×10¹⁰拷贝;普通PCR检出GAPDH的下限为2.0×10⁶拷贝,线性范围:2.0×10⁷～2.0×10⁹拷贝.结论 Taqman荧光定量PCR法比SYBR Green Ⅰ荧光定量PCR法和普通PCR法的灵敏度高,线性范围宽,是核酸水平对GAPDH定量的好方法.

关键词: PCR, GAPDH, 荧光定量

Abstract: Objective In order to find out the best method for GAPDH quantification,the levels of housekeeping gene GAPDH mRNA were detected by Taqman fluorescence quantitative polymerase chain reaction(FQ-PCR),SYBR Green I FQ-PCR and conventional PCR,respectively.Methods GAPDH plasmids were constructed by Blue-White Clone method and checked by PCR and EcoR I.One of the correct clones was selected for isolating a large amount of plasmids.The selected plasmids were then 10-fold diluted and served as standard.Standard curves were prepared using the data from Taqman FQ-PCR,SYBR Green I FQ-PCR and conventional PCR,respectively.The sensitivity and linear range of the three methods were then analyzed.Results The detectable thresholds for Taqman FQ-PCR,SYBR Green I FQ-PCR and conventional PCR were 2.0×10⁴,2.0×10⁷ and 2.0×10⁶ DNA copies,respectively.The linear range for these three methods were 2.0×10⁴～2.0×10¹⁰ copies,2.0×10⁷～2.0×10¹⁰ copies and 2.0×10⁷～2.0×10⁹ DNA copies,respectively.Conclusion Taqman FQ-PCR is a better method for quantifying housekeeping gene GAPDH mRNA than SYBR Green I FQ-PCR and conventional PCR.

Key words: polymerase chain reaction, GAPDH, fluorescence quantification

中图分类号:

Q503

王萍;丛敏;李忆梅;唐淑珍;刘晓明;王宝恩;贾继东;尤红. Taqman荧光定量PCR是核酸水平对管家基因GAPDH进行定量的好方法[J]. 首都医科大学学报, 2006, 27(2): 210-213.

Wang Ping;Cong Min;Li Yimei;Tang Shuzhen;Liu Xiaoming;Wang Baoen;Jia Jidong;You Hong. Taqman Fluorescence Quantitative Polymerase Chain Reaction: A Good Method for Housekeeping Gene GAPDH Quantification[J]. Journal of Capital Medical University, 2006, 27(2): 210-213.

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