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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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C. Kutter, H. Schob, M. Stadler, F. Meins Jr., and A. Si-Ammour
MicroRNA-Mediated Regulation of Stomatal Development in Arabidopsis
PLANT CELL, August 1, 2007; 19(8): 2417 - 2429.
[Abstract] [Full Text] [PDF]

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Y.-R. J. Lee, Y. Li, and B. Liu
Two Arabidopsis Phragmoplast-Associated Kinesins Play a Critical Role in Cytokinesis during Male Gametogenesis
PLANT CELL, August 1, 2007; 19(8): 2595 - 2605.
[Abstract] [Full Text] [PDF]

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A. Kusnierczyk, P. Winge, H. Midelfart, W. S. Armbruster, J. T. Rossiter, and A. M. Bones
Transcriptional responses of Arabidopsis thaliana ecotypes with different glucosinolate profiles after attack by polyphagous Myzus persicae and oligophagous Brevicoryne brassicae
J. Exp. Bot., July 11, 2007; (2007) erm043v2.
[Abstract] [Full Text] [PDF]

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S. Takahashi, H. Bauwe, and M. Badger
Impairment of the Photorespiratory Pathway Accelerates Photoinhibition of Photosystem II by Suppression of Repair But Not Acceleration of Damage Processes in Arabidopsis
Plant Physiology, May 1, 2007; 144(1): 487 - 494.
[Abstract] [Full Text] [PDF]

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E. Oh, S. Yamaguchi, J. Hu, J. Yusuke, B. Jung, I. Paik, H.-S. Lee, T.-p. Sun, Y. Kamiya, and G. Choi
PIL5, a Phytochrome-Interacting bHLH Protein, Regulates Gibberellin Responsiveness by Binding Directly to the GAI and RGA Promoters in Arabidopsis Seeds
PLANT CELL, April 1, 2007; 19(4): 1192 - 1208.
[Abstract] [Full Text] [PDF]

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P. C. Bethke, I. G.L. Libourel, N. Aoyama, Y.-Y. Chung, D. W. Still, and R. L. Jones
The Arabidopsis Aleurone Layer Responds to Nitric Oxide, Gibberellin, and Abscisic Acid and Is Sufficient and Necessary for Seed Dormancy
Plant Physiology, March 1, 2007; 143(3): 1173 - 1188.
[Abstract] [Full Text] [PDF]

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R. Muller, M. Morant, H. Jarmer, L. Nilsson, and T. H. Nielsen
Genome-Wide Analysis of the Arabidopsis Leaf Transcriptome Reveals Interaction of Phosphate and Sugar Metabolism
Plant Physiology, January 1, 2007; 143(1): 156 - 171.
[Abstract] [Full Text] [PDF]

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M. Chen, G. Han, C. R. Dietrich, T. M. Dunn, and E. B. Cahoon
The Essential Nature of Sphingolipids in Plants as Revealed by the Functional Identification and Characterization of the Arabidopsis LCB1 Subunit of Serine Palmitoyltransferase
PLANT CELL, December 1, 2006; 18(12): 3576 - 3593.
[Abstract] [Full Text] [PDF]

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J. Griffiths, K. Murase, I. Rieu, R. Zentella, Z.-L. Zhang, S. J. Powers, F. Gong, A. L. Phillips, P. Hedden, T.-p. Sun, et al.
Genetic Characterization and Functional Analysis of the GID1 Gibberellin Receptors in Arabidopsis
PLANT CELL, December 1, 2006; 18(12): 3399 - 3414.
[Abstract] [Full Text] [PDF]

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R. Bari, B. Datt Pant, M. Stitt, and W.-R. Scheible
PHO2, MicroRNA399, and PHR1 Define a Phosphate-Signaling Pathway in Plants
Plant Physiology, July 1, 2006; 141(3): 988 - 999.
[Abstract] [Full Text] [PDF]


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