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Plant Physiology 139:5-17 (2005)
© 2005 American Society of Plant Biologists

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GENOME ANALYSIS

Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis1,[w]

Tomasz Czechowski, Mark Stitt, Thomas Altmann, Michael K. Udvardi and Wolf-Rüdiger Scheible*

Max-Planck Institute of Molecular Plant Physiology, 14476 Potsdam, Germany

Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future.

1 This work was supported by the Max-Planck Society and the Bundesministerium für Bildung und Forschung-funded project GABI Verbund Arabidopsis III Gauntlets (Carbon and Nutrient Signaling: Test Systems, and Metabolite and Transcript Profiles; 0312277A).

[w] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.105.063743.

* Corresponding author; e-mail scheible{at}mpimp-golm.mpg.de; fax 49–331–567–8136.

Received April 4, 2005; returned for revision May 9, 2005; accepted June 2, 2005.




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