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Validating internal controls for quantitative plant gene expression studies.

ABSTRACT: BACKGROUND: Real-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. RESULTS: Using 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. CONCLUSION: Our 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.

SUBMITTER: Brunner AM

PROVIDER: S-EPMC515301 | biostudies-literature | 2004 Aug

REPOSITORIES: biostudies-literature

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Validating internal controls for quantitative plant gene expression studies.

Brunner Amy M AM Yakovlev Igor A IA Strauss Steven H SH

BMC plant biology 20040818

<h4>Background</h4>Real-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 a ...[more]

PMID: 15317655

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Project description:Prunus rootstock belonging to subgenera Amygdalus (peach), Prunus (plum) and Cerasus (cherry) are either from the same species as the scion or another one. The number of inter-species (including inter-subgenera) hybrids has increased as a result of broadening the genetic basis for stress (biotic and abiotic) resistance/tolerance. Identifying genes associated with important traits and responses requires expression analysis. Relative quantification is the simplest and most popular alternative, which requires reference genes (housekeeping) to normalize RT-qPCR data. However, there is a scarcity of validated housekeeping genes for hybrid Prunus rootstock species. This research aims to increase the number of housekeeping genes suitable for Prunus rootstock expression analysis. Twenty-one candidate housekeeping genes were pre-selected from previous RNAseq data that compared the response of root transcriptomes of two rootstocks subgenera to hypoxia treatment, 'Mariana 2624' (P. cerasifera Ehrh.× P. munsoniana W. Wight & Hedrick), and 'Mazzard F12/1' (P. avium L.). Representing groups of low, intermediate or high levels of expression, the genes were assayed by RT-qPCR at 72 hours of hypoxia treatment and analyzed with NormFinder software. A sub-set of seven housekeeping genes that presented the highest level of stability were selected, two with low levels of expression (Unknown 3, Unknown 7) and five with medium levels (GTB 1, TUA 3, ATPase P, PRT 6, RP II). The stability of these genes was evaluated under different stress conditions, cold and heat with the hybrid 'Mariana 2624' and N nutrition with the hybrids 'Colt' (P. avium × P. pseudocerasus Lindl.) and 'Garnem' [P. dulcis Mill.× (P. persica L.× P. davidiana Carr.)]. The algorithms of geNorm and BestKeeper software also were used to analyze the performance of these genes as housekeepers. Stability rankings varied according to treatments, genotypes and the software for evaluation, but the gene GBT 1 often had the highest ranking. However, most of the genes are suitable depending on the stressor and/or genotype to be evaluated. No optimal number of reference genes could be determined with geNorm software when all conditions and genotypes were considered. These results strongly suggest that relative RT-qPCR should be analyzed separately with their respective best housekeeper according to the treatment and/or genotypes in Prunus spp. rootstocks.

Project description:Abiotic stress, such as drought and high salinity, activates a network of signaling cascades that lead to the expression of many stress-responsive genes in plants. The Arabidopsis FIERY1 (FRY1) protein is a negative regulator of stress and abscisic acid (ABA) signaling and exhibits both an inositol polyphosphatase and a 3',5'-bisphosphate nucleotidase activity in vitro. The FRY1 nucleotidase degrades the sulfation byproduct 3'-phosphoadenosine-5'-phosphate (PAP), yet its in vivo functions and particularly its roles in stress gene regulation remain unclear. Here we developed a LC-MS/MS method to quantitatively measure PAP levels in plants and investigated the roles of this nucleotidase activity in stress response and plant development. It was found that PAP level was tightly controlled in plants and did not accumulate to any significant level either under normal conditions or under NaCl, LiCl, cold, or ABA treatments. In contrast, high levels of PAP were detected in multiple mutant alleles of FRY1 but not in mutants of other FRY1 family members, indicating that FRY1 is the major enzyme that hydrolyzes PAP in vivo. By genetically reducing PAP levels in fry1 mutants either through overexpression of a yeast PAP nucleotidase or by generating a triple mutant of fry1 apk1 apk2 that is defective in the biosynthesis of the PAP precursor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), we demonstrated that the developmental defects and superinduction of stress-responsive genes in fry1 mutants correlate with PAP accumulation in planta. We also found that the hypersensitive stress gene regulation in fry1 requires ABH1 but not ABI1, two other negative regulators in ABA signaling pathways. Unlike in yeast, however, FRY1 overexpression in Arabidopsis could not enhance salt tolerance. Taken together, our results demonstrate that PAP is critical for stress gene regulation and plant development, yet the FRY1 nucleotidase that catabolizes PAP may not be an in vivo salt toxicity target in Arabidopsis.

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Validating internal controls for quantitative plant gene expression studies.

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Validating internal controls for quantitative plant gene expression studies.

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