Selection and Validation of Reference Genes for Quantitative RT-PCR Analysis of Gene Expression in Populus trichocarpa
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- 1College of Life Sciences, Shanxi Normal University, Linfen 041000, China;
2Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China
Received date: 2013-01-10
Revised date: 2013-03-15
Online published: 2013-09-26
Abstract
Quantitative RT-PCR (qRT-PCR) has been widely used in gene expression analysis because of its sensitivity, specificity, and reproducibility. Application of suitable reference genes to normalize qRT-PCR data is critical in analyzing PCR results. We analyzed the expression patterns of 7 housekeeping genes, including TUA8, TUB6, ubiquitin, GAPDH, actin, 18S rRNA and EF1α, in various tissues of greenhouse-grown Populus trichocarpa and Zn-treated in vitro plantlets. The stability of housekeeping gene expression was analyzed with use of 3 software packages, including geNorm, Norm- Finder, and BestKeeper. The genes actin, ubiquitin, EF1α and 18S rRNA were suitable reference genes for efficient normalization of qRT-PCR data, whereas TUB6 and GAPDH were not suitable for analysis of greenhouse-grown plants and Zn-treated plantlets, respectively. These findings were confirmed by comparative profiling of 4 P. trichocarpa NAC genes. This study provides useful information for reference gene selection in qRT-PCR analysis of gene expression in P. trichocarpa. It is also helpful to elucidate the function of P. trichocarpa NAC genes.
Cite this article
Xiaojuan Su, Baoguo Fan, Lichai Yuan, Xiuna Cui, Shanfa Lu . Selection and Validation of Reference Genes for Quantitative RT-PCR Analysis of Gene Expression in Populus trichocarpa[J]. Chinese Bulletin of Botany, 2013 , 48(5) : 507 -518 . DOI: 10.3724/SP.J.1259.2013.00507
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Zhou B, Cao C, Liu C X (2007). Advances in research on translation elongation factor 1 alpha. Lett Biotechnol 18, 281-284.
Bustin S A (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25, 169-193.
Bustin S A (2002). Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29, 23-39.
Bustin S A, Benes V, Nolan T, Pfaffl M W (2005). Quantitative real-time RT-PCR–a perspective. J Mol Endocrinol 34, 597-601.
Basa B, Solti A, Sarvari E, Tamas L (2009). Housekeeping gene selection in poplar plants under Cd-stress: comparative study for real-time PCR normalisation. Funct Plant Biol 36, 1079-1087.
Boava L P, Laia M L, Jacob T R, Dabbas K M, Goncalves J F, Ferro J A, Ferro M I , Furtado E L (2010). Selection of endogenous genes for gene expression studies in Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using RT-qPCR. BMC Res Notes 3, 43.
Carvalho K, de Campos M K, Pereira LF, Vieira L G (2010). Reference gene selection for real-time quantitative polymerase chain reaction normalization in‘‘Swingle’’ citrumelo under drought stress. Anal Biochem 402, 197-199.
Chen X, Truksa M, Shah S, Weselake RJ (2010). A survey of quantitative real-time polymerase chain reaction internal reference genes for expression studies in Brassica napus. Anal Biochem 405, 138-140.
Dheda K, Huggett J F, Bustin S A, Johnson MA, Rook G, Zumla A (2004). Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37, 112-119.
Han X, Lu M, Chen Y, et al (2012). Selection of reliable reference genes for gene expression studies using real-time PCR in tung tree during seed development. PLoS One 7, e43084.
Hochstrasser M (2000). Evolution and function of ubiquitin-like protein-conjugation systems. Nat Cell Biol 2, E153-157.
Hong S Y, Seo P J, Yang M S, Xiang F N, Park C M (2008). Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol 8, 1-11.
Hu R, Qi G, Kong Y Z, Kong D J, G Q, Zhou G K (2010). Comprehensive analysis of NAC domain transcription factor gene family in Populus trichocarpa. BMC Plant Biol 10, 145.
Huis R, Hawkins S, Neutelings G (2010). Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biol 10, 71.
Kim B R, Nam H Y, Kim S U, Kim S L, Chang Y J (2003). Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnol Lett 25, 1869-1872.
Lovdal T, Lillo C (2009). Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Anal Biochem 387, 238-242.
Lu S, Sun Y H, Chiang V L (2008). Stress-responsive microRNAs in Populus. Plant J 55, 131-151.
Luo H, Chen S, Wan H, Chen F, Gu C, Liu Z (2010). Candidate reference genes for gene expression studies in water lily. Anal biochem 404, 100-102.
Mafra V, Kubo K S, Alves-Ferreira M, Ribeiro-Alves M, Stuart R M, Boava L P, Rodrigues C M, Machado M A (2012). Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions. PLoS One 7, e31263.
Mallona I, Lischewski S, Weiss J, Hause B, Egea-Cortines (2010). Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biol 10, 4.
Maroufi A, Bockstaele E Y, Loose M D, (2010). Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11, 15-26.
Martin R C, Hollenbeck V G, Dombrowski J E (2008). Evaluation of reference genes for quantitative RT-PCR in Lolium perenne. Crop Sci 48, 1881-1887.
Mehdi Khanlou K, Van Bockstaele E (2012). A critique of widely used normalization software tools and an alternative method to identify reliable reference genes in red clover (Trifolium pratense L.). Planta 236, 1381-1393.
Nolan T, Hands R E, Bustin S A (2006). Quantification of mRNA using real-time RT-PCR. Nat Protoc 1, 1559-1582.
Pettengill E A, Parmentier-Line C, Coleman G D (2012). Evaluation of qPCR reference genes in two genotypes of Populus for use in photoperiod and low-temperature studies. BMC Res Notes 5, 366.
Pfaffl M W, 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.
Ponton F, Chapuis M P, Pernice M, Sword G A, Simpson S J (2011). Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. J Insect Physiol 57, 840-850.
Regier N, Frey B (2010). Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar. BMC Mol Biol 11, 57.
Reid K E, Olsson N, Schlosser J, Peng F, Lund S T (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, 27–37.
Santella L, Chun J T (2011). Actin, more than just a housekeeping protein at the scene of fertilization. Sci China Life Sci 54, 733-743.
Schmittgen T, Zakrajsek B A (2000). Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46, 69-81.
Suzuki T, Higgins P J, Crawford D R (2000). Control selection for RNA quantitation. BioTechniques 29, 332-337.
Thellin O, Zorzi W, Lakaye B, Borman B D, Coumans B, Hennen G, Grisar T, Lgout A, Heinen E (1999). Housekeeping genes as internal standards: use and limits. J Biotechnol 75, 291-295.
Udvardi M K, Czechowski T, Scheible W R (2008). Eleven golden rules of quantitative RT-PCR. J Plant Cell 20, 1736-1737.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van RoyN, 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, RESEARCH0034.
VanGuilder H D, Vrana K E, Freeman W M (2008). Twenty-five years of quantitative PCR for gene expression analysis. BioTechniques 44, 619-626.
Wan H, Zhao Z, Qian C, Sui Y, Malik AA, Chen J (2010). Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399, 257-261.
Warrington J A, Nair A, Mahadevappa M, Tsyganskaya M (2000). Comparison of human adult and fetal expression and identification of 535 housekeeping maintenance genes. Physiol Genomics 2, 143-147.
Xu M, Zhang B, Su X, et al (2011). Reference gene selection for quantitative real-time polymerase chain reaction in Populus. Anal Biochem 408, 337-339.
Yang Y, Hou S, Cui G, et al (2010). Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Salvia miltiorrhiza. Mol Biol Rep 37, 507-513.
Zhou B, Cao C, Liu C X (2007). Advances in research on translation elongation factor 1 alpha. Lett Biotechnol 18, 281-284.