以梅花鹿P21基因为模板比较相对和绝对荧光定量PCR的准确性  

Comparative Studies between Relative and Absolute PCR Value Using Sika Deer P21 Gene as an Example

作　　者：王大涛[1] 郭倩倩[1] 朱宏伟[1] 刘振[1] 王桂武[1] 李春义[1] 

机构地区：[1]中国农业科学院特产研究所,长春130112

出　　处：《特产研究》2013年第4期10-13,共4页Special Wild Economic Animal and Plant Research

基　　金：973前期专项(2011CB111515);国家自然科学基金(31170950);吉林省自然科学基金(201115129)

摘　　要：实时荧光定量PCR是具有高度灵敏度和特异性的核酸分析技术,以SYBRGreen染料法为基础的相对定量和绝对定量检测方法,由于操作简单、成本较低而倍受青睐。本试验以梅花鹿P21基因为待检测基因,以β-actin作为内参基因,对不同浓度稀释的模板进行相对实时荧光定量PCR检测,然后分别对P21基因和β-actin基因进行绝对定量检测。结果表明,绝对定量的结果比相对定量结果更准确可靠,相对定量在模板256倍稀释后出现较大偏差,在模板浓度较低(低于1 000拷贝)时2种方法都会出现较大误差。因此,建议尽量用绝对定量检测目的基因,当模板浓度太低时建议对样品进行浓缩处理。Real time fluorescent quantitative PCR is a highly sensitive and specific nucleic acid analysis technique, relative and absolute quan- tifications using SYBR Green dye are used widely because of simple operation and low cost. In this study,we detected P21 gene of silo deer using qPCR and β- actin gene was used as an internal control. Samples were diluted to different concentrations and the relative and absolute values of P21 and β - actin genes were determined separately. The results showed that overall absolute quantification was more accurate and more reliable compared to the relative quantification, as the relative quantification showed a large deviation at x 256. Both methods showed big deviations when the template concentration was very low（lower than 1 000 copes）. Therefore, we suggest that using absolute quantification whenever possible,and concentrating samples when the copes target genes are too low.

关 键 词：实时荧光定量PCR 相对定量 绝对定量 P21 

分 类 号：Q781[生物学—分子生物学]