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Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations

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Abstract

Accurate quantification by real-time RT-PCR relies on normalisation of the measured gene expression data. Normalisation with multiple reference genes is becoming the standard, but the best reference genes for gene expression studies within one organism may depend on the applied treatments or the organs and tissues studied. Ideally, reference genes should be evaluated in all experimental systems. A number of candidate reference genes for Arabidopsis have been proposed, which can be used as a starting point to evaluate their expression stability in individual experimental systems by available computer algorithms like geNorm and NormFinder. Using this approach, we identified the best three reference genes from a set of ten candidates, which included three traditional “housekeeping” genes, for normalisation of gene expression when roots and leaves of Arabidopsis thaliana are exposed to cadmium (Cd) and copper (Cu). The expression stabilities of AT5G15710 (F-box protein), AT2G28390 (SAND family protein) and AT5G08290 (mitosis protein YLS8) were the highest when considering the effect to the roots and shoots of Cd and Cu treatments. Even though the effect of Cd and excess Cu on the plants is very different, the same best reference genes were identified when considering Cd or Cu treatments separately. This suggests that these three genes may also be suitable when studying the gene expression after exposure of Arabidopsis thaliana to increased concentrations of other metals.

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Abbreviations

Cd:

Cadmium

Cu:

Copper

ROS:

Reactive oxygen species

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Acknowledgments

This study was funded by a Research Foundation-Flanders grant (project G.0436.06). Tony Remans is a post-doctoral research fellow of the Research Foundation-Flanders (FWO). Karen Smeets was supported by a PhD grant from Hasselt University (tUl-impulsfinanciering-toxicology). We also thank Carine Put and Ann Wijgaerts for their technical assistance.

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Correspondence to Tony Remans.

Electronic supplementary material

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425_2008_706_MOESM1_ESM.pdf

Average expression stability (M) and pairwise variation (V) graphs obtained from the geNorm algorithm using input of all samples (biological replicates of each treatment/organ combination entered separately). (PDF 29 kb)

425_2008_706_MOESM2_ESM.pdf

Average expression stability (M) and pairwise variation (V) graphs obtained from the geNorm algorithm using averaged input per treatment/organ combination for different data sets. (PDF 29 kb)

425_2008_706_MOESM3_ESM.pdf

"NormFinder" output for the selected data sets, obtained by using expression values for individual samples as input (A), or by using an averaged input per treatment/organ combination (B). (PDF 12 kb)

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Remans, T., Smeets, K., Opdenakker, K. et al. Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta 227, 1343–1349 (2008). https://doi.org/10.1007/s00425-008-0706-4

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  • DOI: https://doi.org/10.1007/s00425-008-0706-4

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