Skip to main content
SpringerLink
Log in

Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

Reverse transcription quantitative real-time polymerase chain reaction is the most accurate measure of gene expression in biological systems. The data are analyzed through a process called normalization. Internal standards are essential for determining the relative gene expression in different samples. For this purpose, reference genes are selected based on their constitutive expression across samples. At present, there has not yet been any reference gene identified in any organism that is universally optimal across different tissue types or disease situations. Our goal was to test the regulation of 11 potential references for pea. These included eight commonly used and three new candidates. Twenty-six samples, including different tissues, treatments and genotypes, were addressed in this analysis. For reliable data normalization, the most suitable combination of reference genes in each experimental set was constructed with at least two out the five more stably expressed references in the whole experimental series (i.e. protein phosphatase 2A, β-tubulin, GH720838, actin and GH720808). To validate the determined measure of gene-stability, the gene-specific variation was calculated using different normalization factors. The most non-specific variation was removed when the most stable genes were used, highlighting the importance of the adequate choice of internal controls in gene expression experiments. The set of reference genes presented here will provide useful guidelines as starting point for reference gene selection in pea studies under conditions other than those tested here.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

C q :

Quantification cycle

CV:

Coefficient of variation

E :

PCR amplification efficiency

EF-1α:

Elongation factor 1α

GAPDH :

Glyceraldehyde-3-phosphate dehydrogenase

PLC :

Phospholipase

PP2A :

Protein phosphatase 2A

qPCR:

Quantitative PCR

NF:

Normalization factor

RQ:

Relative quantity

RT:

Reverse transcription

SD:

Standard deviation

SE:

Standard error

References

  • 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 (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193

    Article  PubMed  CAS  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, Huguett J, Kubista M, Mueller R et al (2009) The MIQE Guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  PubMed  CAS  Google Scholar 

  • Choi HK, Mun JH, Kim DJ, Uhm T, Zhu H, Baek JM, Mudge J et al (2004) Estimating genome conservation between crop and model legume species. Proc Natl Acad Sci 101:15289–15294

    Article  PubMed  CAS  Google Scholar 

  • Cruz F, Kalaoun S, Nobile P, Colombo C, Almeida J, Barros L, Romano E et al (2009) Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR. Mol Breeding 23:607–616

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Die JV, Román B, Nadal S, Dita MA, González-Verdejo CI (2009) Expression analysis of Pisum sativum putative defence genes during Orobanche crenata infection. Crop Pasture Sci 60:490–498

    Article  CAS  Google Scholar 

  • Dombrowski J, Martin R (2009) Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress. Plant Sci 176:390–396

    Article  CAS  Google Scholar 

  • Expósito-Rodríguez M, Borges AA, Borges-Pérez A, Pérez JA (2008) Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol 8:131

    Article  PubMed  Google Scholar 

  • FAOSTAT data (2008) http://faostat.fao.org/. Accessed 9 March 2010

  • Guénin 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

    Article  PubMed  Google Scholar 

  • Gutierrez L, Mauriat M, Guénin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C et al (2008a) 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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:R19

    Article  PubMed  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • Hu R, Fan C, Li H, Zhang Q, Fu Y-F (2009) Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol Biol 10:93

    Article  PubMed  Google Scholar 

  • Hugget J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6:279–284

    Article  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Kakar K, Wandrey M, Czechowski T, Gaertner T, Scheible W-R, Stitt M, Torres-Jerez I et al (2008) A community resource for high-throughput quantitative RT-PCR analysis of transcription factor gene expression in Medicago truncatula. Plant Methods 4:18

    Article  PubMed  Google Scholar 

  • Kim B, Nam H, Kim S, Chang YJ (2003) Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnol Lett 25:1869–1872

    Article  PubMed  CAS  Google Scholar 

  • Libault M, Thibivilliers S, Bilgin DD, Radwan O, Benitez M, Clough SJ, Stacey G (2008) Identification of four soybean reference genes for gene expression normalization. Plant Genome 1:44–54

    Article  CAS  Google Scholar 

  • Martin RC, Hollenbeck VG, Dombrowski JE (2008) Evaluation of reference genes for quantitative RT-PCR in Lolium perenne. Crop Sci 48:1881–1887

    Article  CAS  Google Scholar 

  • 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 Bot 56:2907–2914

    Article  PubMed  CAS  Google Scholar 

  • Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582

    Article  PubMed  CAS  Google Scholar 

  • Paolacci AR, Tanzarella OA, Porceddu EP, 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 

  • Ramakers C, Ruijter JM, Deprez RH, Moorman AF (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 13:62–66

    Article  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Rubiales D, Moreno MT, Sillero JC (2005) Search for resistance to crenate broomrape (Orobanche crenata) in pea germplasm. Gen Res Crop Evol 52:853–861

    Article  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Samuel Roberts Noble Foundation (2006) Medicago truncatula Handbook. In: Mathesius U, Journet EP, Sumner LW (eds) http://www.noble.org/MedicagoHandbook/. Accessed 9 March 2010

  • Silveira ED, Alves-Ferreira M, Guimarães LA, Rodrigues da Silva F, Carneiro V (2009) Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha. BMC Plant Biol 9:84

    Article  PubMed  Google Scholar 

  • Tong Z, Gao Z, 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

    Article  PubMed  Google Scholar 

  • Tu L, Zhang X, Liu D, Jin S, Cao J, Zhu L, Deng F, Tan J, Zhang C (2007) Suitable internal control genes for qRT-PCR normalization in cotton fiber development and somatic embryogenesis. Chin Sci Bull 52:3110–3117

    Article  CAS  Google Scholar 

  • Udvardi MK, Czechowski T, Scheible W-R (2008) Eleven golden rules of quantitative RT-PCR. Plant Cell 20:1736–1737

    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

    Article  PubMed  Google Scholar 

  • Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. BioTechniques 39:75–85

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Financial support was provided by the Spanish project RTA2007-00009. J.V. D and C.I. G-V are researchers funded by the ‘Juan de la Cierva’ programme of the Spanish MICINN. The authors acknowledge the USDA-ARS (Washington State University) for the supply of Ps624 seeds.

Author information

Author notes

  1. José V. Die

    Present address: Mejora Genética Vegetal, Instituto de Agricultura Sostenible, Consejo Superior de Investigaciones Científicas, Apdo. 4084, 14080, Córdoba, Spain

Authors and Affiliations

  1. Mejora y Biotecnología, IFAPA “Alameda del Obispo”, 14080, Córdoba, Spain

    José V. Die, Belén Román & Clara I. González-Verdejo

  2. Producción Agraria, IFAPA “Alameda del Obispo”, 14080, Córdoba, Spain

    Salvador Nadal

Authors
  1. José V. Die
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Belén Román
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Salvador Nadal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Clara I. González-Verdejo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to José V. Die.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 906 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Die, J.V., Román, B., Nadal, S. et al. Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta 232, 145–153 (2010). https://doi.org/10.1007/s00425-010-1158-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-010-1158-1

Keywords

Search

Navigation