| 1. |
- Guenin, Stephanie, et al.
(författare)
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Normalization of qRT-PCR data : the necessity of adopting a systematic, experimental conditions-specific, validation of references
- 2009
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Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 60:2, s. 487-493
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Tidskriftsartikel (refereegranskat)abstract
- Quantitative RT-PCR (reverse transcription polymerase chain reaction, also known as qRT-PCR or real-time RT-PCR) has been used in large proportions of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes but, surprisingly, this robust approach is under-utilized in plants. Instead, putative ’housekeeping’ genes tend to be used as references without any proper validation. The concept of normalization in transcript quantification is introduced here and the factors affecting its reliability in qRT-PCR are discussed in an attempt to convince molecular biologists, and non-specialists, that systematic validation of reference genes is essential for producing accurate, reliable data in qRT-PCR analyses, and thus should be an integral component of them.
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| 2. |
- Gutierrez, Laurent, et al.
(författare)
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Combined networks regulating seed maturation
- 2007
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Ingår i: Trends in Plant Science. - : Elsevier BV. - 1360-1385 .- 1878-4372. ; 12:7, s. 294-300
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Forskningsöversikt (refereegranskat)abstract
- Seed maturation is an important phase of seed development during which embryo growth ceases, storage products accumulate, the protective tegument differentiates and tolerance to desiccation develops, leading to seed dormancy. The spatial and temporal regulation of all these processes requires the concerted action of several signaling pathways that integrate information from genetic programs, and both hormonal and metabolic signals. Recent genetic studies have identified some of the interactions that occur between four master regulators in Arabidopsis, increasing our knowledge of the control of the transcriptional program involved in seed maturation. Moreover, several recent breakthroughs have led to a better understanding of the role of abscisic acid signal modulation and the importance of metabolic regulation in the maternal to filial switch leading to the maturation phase.
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| 3. |
- Gutierrez, Laurent, et al.
(författare)
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The lack of a systematic validation of reference genes : a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants.
- 2008
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7644 .- 1467-7652. ; 6:6
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Tidskriftsartikel (refereegranskat)abstract
- Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.
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