db/db小鼠肝脏中实时定量逆转录PCR内参照基因及标准化方法的评估与整合（英文）  

Evaluation and integration of normalization approaches and internal reference genes in real-time quantitative reverse transcription PCR in liver tissues of db/db mice

作　　者：李开济 田增有 田景瑞 门秀丽 吴静 

机构地区：[1]华北理工大学基础医学院病理生理学系,河北省慢性疾病重点实验室,唐山市慢性病临床基础研究重点实验室,河北唐山063000

出　　处：《中国病理生理杂志》2016年第11期2106-2112,共7页Chinese Journal of Pathophysiology

基　　金：Supported by National Natural Science Foundation of China(No.81370477;No.81370918);Science and Technology Research Outstanding Youth Fund of Hebei College(No.Y2011117);Undergraduate Innovation Project(No.201410081061;No.X2014026)

摘　　要：目的:实时定量逆转录PCR(RT-q PCR)结果的标准化对于保证最终结果的准确性尤其重要。常用的标准化方法包括用加入的核酸量校正(ΔCt法)、用单个内参照基因校正(ΔΔCt法)和用统计学软件计算多个内参照基因的几何平均值进行校正。我们在db/db小鼠肝脏中对各种校正方法进行评估。方法:用ge Norm和NormFinder两种软件评估ACTB、e IF5、GAPDH、HMBS、HPRT1、Polr2A和RPLP0共7个内参照基因,以肝脏脂质合成相关基因Thrsp、SCD、SREBP1c和FAS作为目的基因。结果:应用ΔCt法,db/db小鼠肝脏中所有目的基因及GAPDH的表达显著升高(P<0.05)。ge Norm计算认为ACTB和HMBS最稳定。Norm Finder计算认为ACTB最稳定,而GAPDH和RPLP0为最佳组合。以单个基因ACTB或RPLP0,以及ACTB与HMBS,或GAPDH与RPLP0的几何平均值进行校正,db/db小鼠肝脏中除SREBP1c以外,Thrsp、SCD1和FAS的表达均升高(P<0.05)。结论:用ΔCt法校正RTq PCR的结果稳定并具有生物学意义。统计学软件ge Norm或Norm Finder应与ΔCt法整合使用。AIM： Normalizing the results of real-time quantitative reverse transcription polymerase chain reaction（ RT-q PCR） is essential for the accuracy of analysis. Commonly used approaches include input nucleic acid standardization（ ΔCt method）,normalization against a single internal reference gene（ ΔΔCt method）,and geometric averaging of multiple reference gene abundance using statistical software. We evaluated these approaches in the liver of db / db mice,a typical model of fatty liver disease. METHODS： Seven reference genes,β-actin（ ACTB）,eukaryotic initiation factor（ e IF） 5,glyceraldehyde-3-phosphate dehydrogenase（ GAPDH）,hydroxymethylbilane synthase（ HMBS）,hypoxanthineguanine phosphoribosyltransferase（ HPRT） 1,polymerase（ RNA） II（ DNA directed） polypeptide A（ Polr2A） and ribosomal protein P〈0（ RPLP〈0）,were evaluated using software of ge Norm and Norm Finder. Hepatic lipogenesis genes,such as thyroid hormone-responsive protein（ Thrsp）,stearoyl-Co A desaturase（ SCD） 1,sterol regulatory element-binding protein（ SREBP） 1c and fatty acid synthase（ FAS）,were used as target genes of interest. RESULTS： The expression levels of all target genes and GAPDH were significantly elevated（ P〈0. 05） in db / db mouse livers by the ΔCt method. ACTB and HMBS were the most stable genes calculated by the software of ge Norm. Norm Finder analysis indicated that ACTB was the most stable gene,and the best combination of 2 genes was GAPDH and RPLP〈0. Normalization against a single internal reference gene of ACTB or RPLP〈0,the geometric mean of ACTB and HMBS,or GAPDH and RPLP〈0 showed similar results that the expression levels of Thrsp,SCD1 and FAS,but not SREBP1 c increased（ P〈0. 05） in the liver of db / db mice.CONCLUSION： The ΔCt approach ensures a meaningful and biologically significant appraisal of gene expression. Use of the software like ge Norm or Norm Finder should be integrated with ΔCt method.

关 键 词：实时定量逆转录PCR 标准化方法 内参照基因 DB/DB小鼠 

分 类 号：R-331[医药卫生] R34