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


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.

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Fig. 1
Fig. 2
Fig. 3


C q :

Quantification cycle


Coefficient of variation

E :

PCR amplification efficiency


Elongation factor 1α


Glyceraldehyde-3-phosphate dehydrogenase



PP2A :

Protein phosphatase 2A


Quantitative PCR


Normalization factor


Relative quantity


Reverse transcription


Standard deviation


Standard error


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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.

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Correspondence to José V. Die.

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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).

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  • Normalization
  • Pea
  • Quantitative PCR
  • Reference genes
  • Stable expression