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Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains

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The stability of candidate reference genes was evaluated in maize landrace varieties and during multiple grain developmental stages to evaluate the expression of carotenoid-related genes by RT-qPCR for application to maize biofortification.

Abstract

Vitamin A deficiency affects millions of children worldwide; therefore, increasing the content of vitamin A precursors in maize grains is of interest. The study of the expression of genes involved in the carotenoid biosynthetic pathway in maize grains has provided useful information for metabolic engineering approaches. However, reliable results using real-time quantitative polymerase chain reaction (RT-qPCR) experiments are dependent on the use of the appropriate reference genes. In this study, we utilized geNorm and NormFinder softwares to identify the most stably expressed candidate reference genes in samples from seven stages of grain development and from eight landrace varieties. The results of the analysis performed using geNorm indicated that tubulin (TUB) and actin (ACT) were the most suitable reference genes among all experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) showed the least stability. The same result was obtained with the NormFinder software. The minimum number of genes required in each experimental condition to normalize the gene expression data was also determined by geNorm. The expression of phytoene synthase gene (PSY1), the first enzyme in the carotenoid biosynthetic pathway, was overestimated when the least stable candidate gene (GAPDH) was used as the internal control instead of the most stable gene pair (ACT + TUB), thus highlighting the importance of validating reference genes before conducting a RT-qPCR experiment to obtain accurate results. This study is the first survey of the stability of genes for use as reference genes to normalize RT-qPCR data from maize landraces during multiple stages of grain development.

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References

  • Aluru M, Xu Y, Guo R, Wang Z, Li S, White W, Wang C, Rodermel S (2008) Generation of transgenic maize with enhanced provitamin A content. J Exp Bot 59:3551–3562

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Berardo N, Mazzinelli G, Valoti P, Lagana P, Redaellij R (2009) Characterization of maize germplasm for the chemical composition of the grain. Agr Food Chem 57:2378–2384

    Article  CAS  Google Scholar 

  • Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4:14

    Article  PubMed  Google Scholar 

  • Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39

    Article  PubMed  CAS  Google Scholar 

  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative realtime PCR experiments. Clin Chem 55:611–622

    Article  PubMed  CAS  Google Scholar 

  • Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11

  • Chen K, Fessehaie A, Arora R (2012) Selection of reference genes for normalizing gene expression during seed priming and germination using qPCr in Zea mays and Spinacia oleracea. Plant Mol Biol Rep 30:478–487

    Article  CAS  Google Scholar 

  • Coker JS, Davies E (2003) Selection of candidate housekeeping controls in tomato plants using EST data. Biotechniques 35:740–748

    PubMed  CAS  Google Scholar 

  • Czechowski T, Stitt M, Altmann 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

    Article  PubMed  CAS  Google Scholar 

  • Derveaux S, Vandersompele J, Hellemans J (2010) How to do successful gene expression analysis using real-time PCR. Methods 50:227–230

    Article  PubMed  CAS  Google Scholar 

  • Fleige S, Walf V, Huch S, Prgomet C, Sehm J, Pfaffl MW (2006) Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnol Lett 28:1601–1613

    Article  PubMed  CAS  Google Scholar 

  • Fraser PD, Bramley PM (2004) The biosynthesis and nutritional uses of carotenoids. Prog Lipid Res 43:228–265

    Article  PubMed  CAS  Google Scholar 

  • Gutierrez L, Mauriat M, Pelloux J, Bellini C, Wuytswinkel UV (2008) Towards a systematic validation of references in real time RT-PCR. Plant Cell 20:1734–1735

    Article  PubMed  CAS  Google Scholar 

  • Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. BBRC 343:646–651

    Google Scholar 

  • Kulcheski FR, Marcelino-Guimaraes FC, Nepomuceno AL, Abdelnoor RV, Margis R (2010) The use of microRNAs as reference genes for quantitative polymerase chain reaction in soybean. Anal Biochem 406:185–192

    Article  PubMed  CAS  Google Scholar 

  • Lei C, Hai-ying Z, Jian-fei K, Jian-guo L, Wang-jin L, Jian-ye C (2011) Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions. Planta 234:377–390

    Article  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-Delta DeltaCT method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Magneschi L, Kudahettige RL, Alpi A, Perata P (2009) Expansin gene expression and anoxic coleoptiles elongation in rice cultivars. J Plant Physiol 166:1576–1580

    Article  PubMed  CAS  Google Scholar 

  • Manoli A, Sturaro A, Trevisan S, Quaggiotti S, Nonis A (2012) Evaluation of candidate reference genes for qPCR in maize. J Plant Physiol 169:807–815

    Article  PubMed  CAS  Google Scholar 

  • Maroufi A, Van Bockstaele E, De Loose M (2010) Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11:15

    Article  PubMed  Google Scholar 

  • Muñoz-Bertomeu J, Cascales-Miñana B, Alaiz M, Segura J, Ros R (2010) A critical role of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in the control of plant metabolism and development. Plant Signal Behav 5:67–69

    Article  PubMed  Google Scholar 

  • Naqvi S, Zhu C, Farre G, Ramessar K, Bassie L, Breitenbach J, Conesa DP, Ros G, Sandmann G, Capell T, Christou P (2009) Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. PNAS 106:7762–7767

    Article  PubMed  CAS  Google Scholar 

  • Paolacci AR, Tanzarella OA, Porceddu E, Ciaffi M (2009) Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Biol 10:11

    Article  PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Qian-Feng L, Samuel S, Ding-Yang Y, Heng-Xiu Y, Ming-Hong G, Qiao-Quan L (2010) Validation of candidate reference genes for the accurate normalization of real-time quantitative RT-PCR data in rice during seed development. Plant Mol Biol Rep 28:49–57

    Article  Google Scholar 

  • Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Bioph Res Co 313:856–862

    Article  CAS  Google Scholar 

  • Vallabhaneni R, Wurtzel ET (2009) Timing and biosynthetic potential for carotenoid accumulation in genetically diverse germplasm of maize. Plant Physiol 150:562–572

    Article  PubMed  CAS  Google Scholar 

  • 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 RESEARCH0034

  • 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

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors gratefully acknowledge the support of Embrapa Temperate Agriculture, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPEG (Fundação de Apoio a Pequisa e Desenvolvimento Agropecuário Edmundo Gastal).

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There are no conflicts of interests to declare.

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Authors and Affiliations

  1. Empresa Brasileira de Pesquisa Agropecuária de Clima Temperado, Rodovia BR 396, Km 78 Postal code 403, Pelotas, RS, CEP 96001-970, Brazil

    Vanessa Galli, Rafael da Silva Messias & Sérgio Delmar dos Anjos e Silva

  2. Universidade Federal Do Rio Grande Do Sul, Centro de Biotecnologia, Av. Bento Gonçalves 9500, Campus do Vale, Porto Alegre, RS, Caixa Postal 15005, CEP 91501-970, Brazil

    Vanessa Galli

  3. Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Campus Universitário s/n, Pelotas, RS, Caixa Postal 354, CEP 96010-900, Brazil

    Cesar Valmor Rombaldi

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  1. Vanessa Galli
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  2. Rafael da Silva Messias
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  3. Sérgio Delmar dos Anjos e Silva
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  4. Cesar Valmor Rombaldi
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Corresponding author

Correspondence to Vanessa Galli.

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Communicated by H. Jones.

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Galli, V., da Silva Messias, R., dos Anjos e Silva, S.D. et al. Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains. Plant Cell Rep 32, 1869–1877 (2013). https://doi.org/10.1007/s00299-013-1499-x

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  • DOI: https://doi.org/10.1007/s00299-013-1499-x

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