Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis

• Published in: Plant physiology (Bethesda) Vol. 139; no. 1; pp. 5 - 17
• Main Authors: Czechowski, Tomasz, Stitt, Mark, Altmann, Thomas, Udvardi, Michael K, Scheible, Wolf-Růdiger
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Biologists 01.09.2005; American Society of Plant Physiologists
• Subjects: analysis; Arabidopsis; Arabidopsis - genetics; Arabidopsis Proteins; Arabidopsis Proteins - genetics; Arabidopsis thaliana; Biological and medical sciences; Complementary DNA; Computer software; Fundamental and applied biological sciences. Psychology; Gene Expression Profiling; Gene Expression Profiling - methods; Gene Expression Profiling - standards; gene expression regulation; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Gene Expression Regulation, Plant - genetics; Genes; Genes, Plant; Genes, Plant - genetics; Genes. Genome; genetics; genome; Genome Analysis; Genome, Plant; Hormones; messenger RNA; methods; microarray technology; Molecular and cellular biology; Molecular genetics; molecular sequence data; nucleotide sequences; Oligonucleotide Array Sequence Analysis; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotide Array Sequence Analysis - standards; plant genetics; Plant Leaves; Plant Leaves - genetics; Plant Roots; Plant Roots - genetics; Plant Shoots; Plant Shoots - genetics; Plants; Polymerase chain reaction; Reference Standards; Reproducibility of Results; reverse transcriptase polymerase chain reaction; RNA; RNA, Messenger; RNA, Messenger - analysis; RNA, Messenger - genetics; RNA, Plant; RNA, Plant - analysis; RNA, Plant - genetics; Seedlings; Sensitivity and Specificity; Series convergence; transcription (genetics); Transcription, Genetic; Transcription, Genetic - genetics; Transcription; Enzyme; Phosphoprotein phosphatase; Phosphoric monoester hydrolases; Esterases; Hybridization; Gene expression; Arabidopsis thaliana; Complementary DNA; Gene; Cruciferae; Dicotyledones; Angiospermae; Development; Hydrolases; Spermatophyta
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.105.063743

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.