董恩妮;梁青;李利;王林杰;仲涛;王永;张红平;
DONG En-ni1,LIANG Qing1,LI Li1,WANG Lin-jie1,ZHONG Tao1,WANG Yong2,ZHANG Hong-ping1*(1.Institute of Animal Genetics and Breeding,Sichuan Agricultural University,Sichuan Ya′an 625014,China; 2.Southwest University for Nationalities,Sichuan Chengdu 610225,China)

• 1:四川农业大学动物遗传育种研究所
• 2:西南民族大学

摘要(Abstract)：

实时荧光定量PCR(qRT-PCR)作为一种检测mRNA的敏感方法已被广泛应用于基因的表达分析。在利用qRT-PCR技术检测目的基因的表达水平时,为了减少检测样本在RNA产量、质量和反转录效率上可能存在的差异,需要选择合适的稳定内参基因作为校正标准,但是迄今为止尚未找到在所有条件下均可稳定表达的内参基因。近年来出现了筛选稳定性好的内参基因的新方法,如使用GeNorm软件、基因芯片数据、EST数据库。本文就qRT-PCR技术中内参基因的意义、参考基因在物种及组织上的表达特异性、内参基因选择方法进行了综述。
As a sensitive method for detection mRNA abundance,the quantitative real-time PCR(qRT-PCR) has been widely used to analyze gene expression.In order to reduce the possible differences in RNA quality and reverse transcription efficiency,it is necessary and important to choose suitable and stable reference genes as calibration standard in qRT-PCR.So far,no single gene which can act as a universal reference gene was reported.However,new methods to select stabilized reference genes have been discovered,such as the use of GeNorm software,gene-chip expression data and public deposited EST data.The present review will discuss about the selection of reference genes in qRT-PCR.

关键词(KeyWords)： 实时荧光定量PCR;内参基因;稳定性
qRT-PCR;reference gene;stability

Abstract:

Keywords:

基金项目(Foundation): 四川省科技支撑计划(2011NZ0003、2011NZ0099-36)

作者(Authors): 董恩妮;梁青;李利;王林杰;仲涛;王永;张红平;
DONG En-ni1,LIANG Qing1,LI Li1,WANG Lin-jie1,ZHONG Tao1,WANG Yong2,ZHANG Hong-ping1*(1.Institute of Animal Genetics and Breeding,Sichuan Agricultural University,Sichuan Ya′an 625014,China; 2.Southwest University for Nationalities,Sichuan Chengdu 610225,China)

参考文献(References)：

• [1]朱玉贤,李毅,郑晓峰.现代分子生物学[M].北京:高等教育出版社,2007.
• [2]Lee E J,Schmittgen T D.Comparison of RNA assay methodsused to normalize cDNA for quantitative real-time PCR[J].AnalBiochem,2006,357(2):299-301.
• [3]Livak K J,Schmittgen T D.Analysis of relative gene expressiondata using real-time quantitative PCR and the2-△△CT method[J].Methods,2001,25(4):402-408.
• [4]Sc hmittgen T D,Livak K J.Analyzing real-time PCR data by thecomparative C(T)method[J].Nat Protoc,2008,3(6):1101-1108.
• [5]胡瑞波,范成明,傅永福.植物实时荧光定量PCR内参基因的选择[J].中国农业科技导报,2009,l1(6):30-36.
• [6]Dheda K,Huggett J F,Bustin S A,et al.Validation of house-keeping genes for normalizing RNA expression in real-time PCR[J].Bio Techniques,2004,37(1):112-119.
• [7]白晓燕,林嘉颖,吴一龙.肺组织标本荧光定量PCR内参基因精确性鉴定[J].肿瘤研究与临床,2010,(3):186-188.
• [8]许强,张婷.乙肝相关肝癌组织实时定量RT-PCR分析中内参基因的选择[J].齐齐哈尔医学院学报,2010,31(18):2857-2861.
• [9]Gilsbach R,Kouta M,Bonisch H,et al.Comparison of in vitroand in vivo reference genes for internal standardization of real-time PCR data[J].Bio Techniques,2006,40:173-177.
• [10]董晓丽,王加启,卜登攀,等.幼龄小鼠小肠组织内参基因的选择[J].甘肃农业大学学报,2009,44(5):20-24.
• [11]董晓丽.小鼠乳腺、肝脏、小肠组织中内参基因稳定性比较[D].江苏:扬州大学,2009.
• [12]台玉磊,韩立强,杨国庆,等.仔猪组织基因表达中实时定量PCR内参基因的选择[J].农业生物技术学报,2010,18(4):732-736.
• [13]Walker C G,Meier S,Mitchell M D,et al.Evaluation of real-timePCR endogenous control genes for analysis of gene expression inbovine endometrium[J].BMC Mol Biol,2009,10(100):1-12.
• [14]P eletto S,Bertuzzi S,Campanella C,et al.Evaluation of internalreference genes for quantitative expression analysis by real-timePCR in ovine whole blood[J].Int J Mol Sci,2011,12:7732-7747.
• [15]Frota I M A,Leitao C C F,Costa J J N,et al.Stability ofhousekeeping genes and expression of locally produced growthfactors and hormone receptors in goat preantral follicles[J].Zygote,2011,19(1):71-83.
• [16]Yin R,Liu X,Liu C,et al.Systematic selection of housekeepinggenes for gene expression normalization in chicken embryofibroblasts infected with Newcastle disease virus[J].Biochem BiophCo,2011,413(4):537-540.
• [17]Boever S D,Vangestel C,Backer P D,et al.Identification andvalidation of housekeeping genes as internal control for geneexpression in an intravenous LPS infiammation model inchickens[J].Vet Immunol Immunop,2008,122(3-4):312-317.
• [18]王忠伟,郭景茹,王建发,等.新生籽鹅组qRT-PCR分析中内参基因的选择[J].中国兽医学报,2012,32(3):427-431.
• [19]Zhong H,Simons W J.Direct comparison of GAPDH,β-actin,cyclophilin,and 28S rRNA as internal standards for quantifyingRNA levels under hypoxia[J].Biochem Bioph Res Co,1999,259(3):523-526.
• [20]Nygard A B,Jorgensen C B,Cirera S,et al.Selection ofreference genes for gene expression studies in pig tissues using SYBR green qPCR[J].BMC Mol Biol,2007,8(67):1-6.
• [21]Tokunaga K,Nakamura Y,Sakata K,et al.Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancers[J].Cancer Res,1987,47:5616-5619.
• [22]Barber R D,Harmer D W,Coleman R A,et al.GAPDH as a housekeeping gene:analysis of GAPDH mRNA expression in a panel of72human tissues[J].Physiol Genomics,2005,21:389-395.
• [23]Glare E M,Divjak M,Baiey M J,et al.β-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels[J].Thorax,2002,57:765-770.
• [24]Hao F,Jia C,Jia N L,et al.Cloning of black carpβ-actin gene and primarily detecting the function of its promoter region[J].Acta Genetica Sinica,2006,33(2):133-140.
• [25]Bas A,Forsberg G,Hammarstrom S,et al.Utility of the housekeeping genes18S rRNA,b-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes[J].Scand J Immunol,2004,59:566-573.
• [26]Lossos I S,Czerwinski D K,Wechser M A,et al.Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies[J].Leukemia,2003,17:789-795.
• [27]周瑞雪,蒙涛,赵发兰,等.草鱼MYH mRNA表达量分析中采用的内参基因稳定性比较[J].基因组学与应用生物学,2009,28(5):896-900.
• [28]Tri caricoa C,Pinzania P,Bianchi S,et al.Quantitative real-time reverse transcription polymerase chain reaction:normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies[J].Anal Biochem,2002,309(2):293-300.
• [29]Schmid H,Cohen C D,Henger A,et al.Validation of endogenous controls for gene expression analysis in microdissected human renal biopsies[J].Kidney Int,2003,64(1):356-360.
• [30]Vandesompele J,Preter D K,Pattyn F,et al.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J].Genome Biol,2002,3(7):1-12.
• [31]Etschmann B,Wilcken B,Stoevesand K,et al.Selection of reference genes for quantitative real-time PCR analysis in canine mammary tumors using the GeNorm algorithm[J].Vet Pathol,2006,43:934-942.
• [32]Boda E,Pini A,Hoxha E,et al.Selection of reference genes for quantitative real-time RT-PCR studies in mouse brain[J].Mol Neurosci,2009,37:238-253.
• [33]Pfaffl M W,Tichopad A,Prgomet C,et al.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity:BestKeeper–Excel-based tool using pair-wise correlations[J].Biotechnol Lett,2004,26:509-515.
• [34]Andersen C L,Jensen K,Qrntoft T F.Normalization of real-time quantitative reverse transcription-PCR data:a model-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets[J].Cancer Res,2004,64(15):5245-5250.