The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants.

By L. Gutierrez, Mélanie Mauriat, Stéphanie Guénin, J. Pelloux, J. Lefebvre, Romain Louvet, C. Rustérucci, T. Moritz, F. Guerineau, C. Bellini and 1 more. Plant Biotechnology Journal, 2008

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Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.

Subfields: Ecology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/8f8626b55872a473851c6e02608128a77cccbeb1

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Citations in the corpus

Identification of suitable reference genes for studying gene expression in cucumber plants subjected to abiotic stress and growth regulators (2011, background): “Some recently published reports have also proved that ACT might not fulfill the requirements of reference gene definition (Gutierrez et al. 2008; Nicot et al. 2005; Tong et al. 2009).”
A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. (2010, methodology): “…Bio-Rad meets all of these criteria and comes packaged with the CFX96 In RT-qPCR experiments, reference genes are used as controls to normalize the data by correcting for differences in quantities of cDNA used as a template (Gutierrez et al. 2008, Huggett et al. 2005, Vandesompele et al. 2002).”
The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. (2009, background): “Unfortunately, although there is anincreasedawarenessoftheimportanceofsystematic validationandalthoughthepotentiallyhighlymisleading effects of the use of inappropriate reference genes for normalization are widely known, these considerations also are still widely disregarded ( 70 ).”