Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress
Top Cited Papers
- 23 January 2010
- journal article
- Published by Springer Science and Business Media LLC in Molecular Genetics and Genomics
- Vol. 283 (3), 233-241
- https://doi.org/10.1007/s00438-010-0511-1
Abstract
Real-time RT-PCR is a powerful technique for the measurement of gene expression, but its accuracy depends on the stability of the internal reference gene(s) used for data normalization. Tobacco (Nicotiana tabacum) is an important model in studies of plant gene expression, but stable reference genes have not been well-studied in the tobacco system. We address this problem by analysing the expression stability of eight potential tobacco reference genes. Primers targeting each gene (18S rRNA, EF-1alpha, Ntubc2, alpha- and beta-tubulin, PP2A, L25 and actin) were developed and optimized. The expression of each gene was then measured by real-time PCR in a diverse set of 22 tobacco cDNA samples derived from developmentally distinct tissues and from plants exposed to several abiotic stresses. L25 and EF-1alpha demonstrated the highest expression stability, followed by Ntubc2. Measurement of L25 and EF-1alpha was sufficient for accurate normalization in either the developmental or stress-treated samples, but Ntubc2 was also required when considering the entire sample set. Analysis of a tobacco circadian gene (NTCP-23) verified these reference genes in an additional context, and all techniques were optimized to enable a high-throughput approach. These results provide a foundation for the more accurate and widespread use of real-time RT-PCR in tobacco.Keywords
This publication has 35 references indexed in Scilit:
- Selection of plastid- and nuclear-encoded reference genes to study the effect of altered endogenous cytokinin content on photosynthesis genes in Nicotiana tabacumPhotosynthesis Research, 2009
- Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stressAnalytical Biochemistry, 2009
- Identification and validation of reference genes for quantitative RT-PCR normalization in wheatBMC Molecular Biology, 2009
- Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of referencesJournal of Experimental Botany, 2009
- Transfer of Plastid DNA to the Nucleus Is Elevated during Male Gametogenesis in TobaccoPlant Physiology, 2008
- A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCRBMC Biotechnology, 2008
- Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development processBMC Plant Biology, 2008
- Standardized determination of real-time PCR efficiency from a single reaction set-upNucleic Acids Research, 2003
- Control Genes and Variability: Absence of Ubiquitous Reference Transcripts in Diverse Mammalian Expression StudiesGenome Research, 2002
- Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT MethodMethods, 2001