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JXB Advance Access published online on September 27, 2005
Journal of Experimental Botany, doi:10.1093/jxb/eri285
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© The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received January 26, 2005
Accepted August 8, 2005

RESEARCH PAPER

Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress

Nathalie Nicot 1, Jean-François Hausman 1, Lucien Hoffmann 1, and Danièle Evers 1*

1 Centre de Recherche Public-Gabriel Lippmann, Environment and Biotechnologies Research Unit, 41 rue du Brill, L-4422 Belvaux, Luxembourg

* To whom correspondence should be addressed.
Danièle Evers, E-mail: evers{at}lippmann.lu


  Abstract

Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. Real-time RT-PCR is at present the most sensitive method for the detection of low abundance mRNA. To avoid bias, real-time RT-PCR is referred to one or several internal control genes, which should not fluctuate during treatments. Here, the non-regulation of seven housekeeping genes ({beta}-tubulin, cyclophilin, actin, elongation factor 1-{alpha} (ef1{alpha}), 18S rRNA, adenine phosphoribosyl transferase (aprt), and cytoplasmic ribosomal protein L2) during biotic (late blight) and abiotic stresses (cold and salt stress) was tested on potato plants using geNorm software. Results from the three experimental conditions indicated that ef1{alpha} was the most stable among the seven tested. The expression of the other housekeeping genes tested varied upon stress. In parallel, a study of the variability of expression of hsp20.2, shown to be implicated in late blight stress, was realized. The relative quantification of the hsp20.2 gene varied according to the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.

Keywords: Abiotic stress; housekeeping genes; late blight; real-time RT-PCR; normalization; Solanum tuberosum.
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