Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/59736
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Type: Journal article
Title: Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress
Author: Schmidt, Gregor W.
Delaney, Sven Kristov
Citation: Molecular Genetics and Genomics, 2010; 283(3):233-241
Publisher: Springer
Issue Date: 2010
ISSN: 1617-4615
School/Discipline: School of Molecular and Biomedical Science
Statement of
Responsibility: 
Gregor W. Schmidt and Sven K. Delaney
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-1α, Ntubc2, α- and β-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-1α demonstrated the highest expression stability, followed by Ntubc2. Measurement of L25 and EF-1α 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: Tobacco; Real-time RT-PCR; Normalization; Internal reference gene; Nicotiana
Rights: © Springer-Verlag 2010
DOI: 10.1007/s00438-010-0511-1
Appears in Collections:Molecular and Biomedical Science publications

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