植物实时荧光定量PCR内参基因的特点及选择

doi:10.3724/SP.J.1259.2012.00427

植物学报 ›› 2012, Vol. 47 ›› Issue (4): 427-436.DOI: 10.3724/SP.J.1259.2012.00427

• 专题论坛 • 上一篇

植物实时荧光定量PCR内参基因的特点及选择

袁伟^1,2, 万红建¹, 杨悦俭^1*

¹浙江省农业科学院蔬菜研究所, 杭州 310021;
²南京农业大学园艺学院, 南京 210095

收稿日期:2011-10-28 修回日期:2012-03-19 出版日期:2012-07-01 发布日期:2012-07-26
通讯作者: 杨悦俭
基金资助:
国家自然科学基金项目;浙江省公益技术研究农业项目;浙江省重大科技专项

Characterization and Selection of Reference Genes for Real-time Quantitative RT-PCR of Plants

Wei Yuan^1,2, Hongjian Wan¹, Yuejian Yang^1*

¹Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

²College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

Received:2011-10-28 Revised:2012-03-19 Online:2012-07-01 Published:2012-07-26
Contact: Yuejian Yang

摘要/Abstract

摘要： 实时荧光定量PCR(qRT-PCR)具有灵敏度高、特异性强、重复的动态定量范围和高通量等优点, 是进行植物基因表达和转录分析最常用的技术手段之一。选择合适的内参基因是正确运用实时荧光定量PCR分析目标基因表达变化的前提。近年来, 大量研究表明, 内参基因的选择应取决于研究者的实验条件; 随着实验条件的变化, 内参基因的选择也随之变化。因此, 实时荧光定量PCR结果分析的准确性在很大程度上依赖于所选择的内参基因是否适合。该文从内参基因的选择、常用内参基因的特点、新内参基因的挖掘、应用内参基因组合的优点和内参基因的稳定性评价等几方面进行综述, 以期为研究者在实验中选择合适的内参基因提供参考和理论依据。

Abstract: Real-time quantitative RT-PCR (qRT-PCR) is one of the most common technologies used for gene expression and transcriptome analysis, with its high sensitivity, specificity, good reproducibility, wide dynamic quantification range and high-throughput capacity. Selecting the appropriate reference genes is the first step in analyzing the expression of genes of interest. Selecting appropriate reference genes depends on experimental conditions, and selection of reference genes changes after the experimental conditions. Therefore, the accuracy of results from qRT-PCR analysis largely depends on the reference genes used. In this paper, we give a comprehensive summary of reference genes for qRT-PCR, including their selection, characteristics of traditional reference genes, mining new reference genes, the advantage of combining different reference genes, and how to assess stable reference gene expression. The results provide a theoretical foundation for selecting appropriate reference genes for qRT-PCR of plants.

袁伟, 万红建, 杨悦俭. 植物实时荧光定量PCR内参基因的特点及选择. 植物学报, 2012, 47(4): 427-436.

Wei Yuan, Hongjian Wan, Yuejian Yang. Characterization and Selection of Reference Genes for Real-time Quantitative RT-PCR of Plants. Chinese Bulletin of Botany, 2012, 47(4): 427-436.

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://www.chinbullbotany.com/CN/10.3724/SP.J.1259.2012.00427

https://www.chinbullbotany.com/CN/Y2012/V47/I4/427

参考文献

陈凤花, 王琳, 胡丽华 (2005). 实时荧光定量RT-PCR内参基因的选择. 临床检验杂志 23, 393-395.
胡瑞波, 范成明, 傅永福 (2009). 植物实时荧光定量PCR内参基因的选择. 中国农业科技导报, 11, 30-36.
Andersen CL, Jensen JL, Orntoft TF (2004). 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. Cancer Res 64, 5245-5250.
Brunner AM, Yakovlev IA, Strauss SH (2004). Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4, 14.
Coker JS, Davis E (2003). Selection of candidate housekeeping controls in tomato plants using EST data. Biotechniques 35, 75-748.
Cruz F, Kalaoun S, Nobile P, Colombo C, Almeida J, Barros L, Romano E, Grossi-de-Sá MF, Vaslin M, Alves-Ferreira M (2009). Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR. Mol Breeding 23, 607-616.
Czechowski T, Stitt M, Altman T, Udvardi MK, Scheible WR (2005). Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139, 5-17.
Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A (2004). Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37, 112-119.
Die JV, Román B, Nadal S, González-Verdejo CI (2010). Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta 232, 145-153.
Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GAW, Zumla A (2005). The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 344, 141-143.
Faccioli P, Ciceri G P, Provero P, Stancad A M, Morcia C, Terzi V (2007). A combined strategy of “in silico” transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies. Plant Mol Biol 63, 679-688.
Gutierrez L, Mauriat M, Guénin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Van Wuytswinkel O (2008). The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol J 6, 609-618.
Wong ML, Medrano JF (2005). Real-time PCR for mRNA quantitation. Biotechniques 39, 75-85.
Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L (2009). Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions specific, validation of references. J Exp Bot 60, 487-493.
Hong SY, Seo PJ, Yang MS, Xiang F, Park CM (2008). Exploring valid reference genes for gene expression studies in Brachypodium distacyon by real-time PCR. BMC Plant Biol 8,112.
Huggett J, Dheda K, Bustin S, Zumla A (2005). Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6, 279-284.
Hu RB, Fan CM, Li HY, Zhang QZ, Fu YF (2009). Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol Biol 10, 93.
Iskandar HM, Simpson RS, Casu RE, Bonnet GD, Maclean DJ, Manners JM (2004). Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene in sugarcane. Plant Mol Biol Rep 22, 325-337.
Jain M (2009). Genome-wide identification of novel internal control genes for normalization of gene expression during various stages of development in rice. Plant Sci 176, 702-706.
Jian B, Liu B, Bi YR, Hou WS, Wu CX, Han TF (2008). Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol 9: 59.
Long XY, Wang JR, Ouellet T, Rocheleau H, Wei YM, Pu ZE, Jiang QT, Lan XJ, Zheng, YL (2010). Genome-wide identification and evaluation of novel internal control genes for Q-PCR based transcript normalization in wheat. Plant Mol Biol 74, 307-311.
Nicot N, Hausman JF, Hoffmann L, Evers D (2005). Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Boty 56, 2907-2914.
Nolan T, Hands RE, Bustin SA (2006). Quantification of mRNA using real-time RT-PCR. Nat Protoc 1, 1559-1582.
Paolacci AR, Tanzarella OA, Porceddu EP, Ciaffi M (2009). Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Biolol 10, 11.
Pfaffl, MW (2006). Real-Time PCR. New York: International University Line. pp. 63-82.
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—excel-based tool using pair-wise correlations. Biotechnol Lett 26, 509-515.
Quackenbush J (2002). Microarray data normalization and transformation. Nat Genet 32, 496-501.
Reid KE, Olsson N, Schlosser J, Peng F, Lund ST (2006). An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol 6, 27.
Remans T, Smeets K, Opdenakker K, Mathijsen D, Vangronsveld J, Cuypers A (2008). Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta 227, 1343-1349.
Schmid H, Cohen CD, Henger A, Irrgang S, Schl?ndorff D, Kretzler M (2003). Validation of endogenous controls for gene expression analysis in micro dissected human renal biopsies. Kidney Int 64, 356-360.
Silver N, Best S, Jiang J, Thein SL (2006). Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7, 33.
Tiziana M, Elisa S, Donato G, Fabrizio C (2010). Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants. Molecular Plant Pathol 11, 805-816.
Tong ZG, Gao ZH, Wang F, Zhou J, Zhang Z (2009). Selection of reliable reference genes for gene expression studies in peach using real time PCR. BMC Mol Biol 10, 71.
Tu LL, Zhang XL, Liu DQ, Jin SX, Cao JL, Zhu LF, Deng FL, Tan JF, Zhang CB (2007). Suitable internal control genes for qRT-PCR normalization in cotton fiber development and somatic embryogenesis. Chinese Sci Bull 52, 3110-3117.
Udvardi MK, Czechowski T, Scheible WR (2008). Eleven golden rules of quantitative RT-PCR. Plant Cell 20, 1736-1737.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, 0034.1-0034.11.
Volkov RA, Panchuk II. Schoffl F (2003). Heat-stress dependency and developmental modulation of gene expression: the potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR. J Exp Bot 54, 2343-2349.
Zhu J, He F, Song S, Wang J, Yu J (2008). How many human genes can be defined as housekeeping with current expression data? BMC Genomics 9, 172.

植物实时荧光定量PCR内参基因的特点及选择

Characterization and Selection of Reference Genes for Real-time Quantitative RT-PCR of Plants

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics

本文评价