Normalisation of real-time RT-PCR gene expression measurements in <Emphasis Type="Italic">Arabidopsis </Emphasis><Emphasis Type="Italic">thaliana</Emphasis> exposed to increased metal concentrations |
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Authors: | Tony Remans Karen Smeets Kelly Opdenakker Dennis Mathijsen Jaco Vangronsveld Ann Cuypers |
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Institution: | (1) Environmental Biology, Hasselt University, Agoralaan D, 3590 Diepenbeek, Belgium |
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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.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | Cadmium Copper Metal stress Reference genes Housekeeping genes Real-time PCR qBase geNorm NormFinder |
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