Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis
Top Cited Papers
- 1 September 2005
- journal article
- Published by Oxford University Press (OUP) in Plant Physiology
- Vol. 139 (1) , 5-17
- https://doi.org/10.1104/pp.105.063743
Abstract
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.Keywords
This publication has 31 references indexed in Scilit:
- Validating internal controls for quantitative plant gene expression studiesBMC Plant Biology, 2004
- Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data SetsCancer Research, 2004
- Validation of housekeeping genes for normalizing RNA expression in real-time PCRBioTechniques, 2004
- Real-time PCR: what relevance to plant studies?Journal of Experimental Botany, 2004
- Real‐time RT‐PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root‐ and shoot‐specific genesThe Plant Journal, 2004
- Selection of candidate housekeeping controls in tomato plants using EST dataBioTechniques, 2003
- A comparison of normalization methods for high density oligonucleotide array data based on variance and biasBioinformatics, 2003
- Expression Profiling of the Whole Arabidopsis Shaggy-Like Kinase Multigene Family by Real-Time Reverse Transcriptase-Polymerase Chain ReactionPlant Physiology, 2002
- Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problemsJournal of Molecular Endocrinology, 2002
- Ribosomal 18S RNA Prevails over Glyceraldehyde-3-Phosphate Dehydrogenase and β-Actin Genes as Internal Standard for Quantitative Comparison of mRNA Levels in Invasive and Noninvasive Human Melanoma Cell SubpopulationsAnalytical Biochemistry, 2001