Validating internal controls for quantitative plant gene expression studies

By A. Brunner, Igor A. Yakovlev, Steven H. Strauss. BMC Plant Biology, 2004

605 citations recorded by the source corpus.

BackgroundReal-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed.ResultsUsing real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied.ConclusionOur results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/a9fe729bce8cfd37650d9b3302219f248ad35028

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Citations in the corpus

Reference Gene Selection for Real-Time Quantitative Polymerase Chain Reaction of mRNA Transcript Levels in Chinese Cabbage (Brassica rapa L. ssp. pekinensis) (2010, background): “…reference gene in plants; the transcript level of this gene was stable over a certain period in eight tissue samples collected from poplar trees (Brunner et al. 2004), and it was considered as the most stable reference gene among the eight candidate genes in drought-stressed leaves and…”
RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization (2011, methodology): “A second approach is to normalize against multiple reference genes and to use appropriate statistical models to improve the selection of genes with minimal variance [8-14].”
Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis1[w] (2005, methodology): “…dehydrogenase (GAPDH), elongation factor-1a (EF-1a), polyubiquitin (UBQ), actin (ACT), and a-tubulin and b-tubulin (TUA and TUB, respectively) genes (Goidin et al., 2001; Bustin, 2002; Kim et al., 2003; Andersen et al., 2004; Brunner et al., 2004; Dheda et al., 2004; Radonić et al., 2004).”
Identification of Four Soybean Reference Genes for Gene Expression Normalization (2008, background): “…potato (Solanum tuberosum L.; Nicot et al., 2005), GAPDH, actin and EF1-α in grape (Vitis spp.; Reid et al., 2006), ubiquitin in poplar (Populus spp.; Brunner et al., 2004), and ubiquitin, EF-1α, 18S and 25S rRNA in rice (Oryza sativa L.; Kim et al., 2003; Jain et al., 2006) are considered as…”
Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR (2009, background): “The importance of reference genes for plant qPCR analysis has been recently emphasized, even though the identification of these genes is quite laborious ( Brunner et al. 2004; Reid et al. 2006).”
Identification of suitable reference genes for studying gene expression in cucumber plants subjected to abiotic stress and growth regulators (2011, background): “2005), poplar (Brunner et al. 2004), potato (Nicot et al.”
Reference genes in real-time PCR (2013, methodology): “In order to use ANOVA model and the F-test assumptions concerning homogeneity of variance and normality of data must be fulfilled (Brunner et al. 2004).”
An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development (2006, positive): “[10] showed that two of their most stable reference genes represented both high and low expression levels compared to the genes being analysed, demonstrating that the level of expression of the reference genes does not affect accuracy.”
Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. (2005, background): “…al., 2004), bacteria and viruses (Stöcher et al., 2002, 2003; Savli et al., 2003), and, as far as is known, only a few have concerned plants, for example , barley (Burton et al., 2004), rice (Kim et al., 2003), poplar (Brunner et al., 2004), Arabidopsis thaliana, and tobacco (Volkov et al., 2003).”
Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. (2006, background): “A UBQ and a TUA gene were most stably expressed in tested tissue samples of poplar (genus Populus) among the 10 reference genes [18].”