Dataset Information

Reference genes for the normalization of gene expression in eucalyptus species.

ABSTRACT: Gene expression analysis is increasingly important in biological research, with reverse transcription-quantitative PCR (RT-qPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Considering the increased sensitivity, reproducibility and large dynamic range of this method, the requirements for proper internal reference gene(s) for relative expression normalization have become much more stringent. Given the increasing interest in the functional genomics of Eucalyptus, we sought to identify and experimentally verify suitable reference genes for the normalization of gene expression associated with the flower, leaf and xylem of six species of the genus. We selected 50 genes that exhibited the least variation in microarrays of E. grandis leaves and xylem, and E. globulus xylem. We further performed the experimental analysis using RT-qPCR for six Eucalyptus species and three different organs/tissues. Employing algorithms geNorm and NormFinder, we assessed the gene expression stability of eight candidate new reference genes. Classic housekeeping genes were also included in the analysis. The stability profiles of candidate genes were in very good agreement. PCR results proved that the expression of novel Eucons04, Eucons08 and Eucons21 genes was the most stable in all Eucalyptus organs/tissues and species studied. We showed that the combination of these genes as references when measuring the expression of a test gene results in more reliable patterns of expression than traditional housekeeping genes. Hence, novel Eucons04, Eucons08 and Eucons21 genes are the best suitable references for the normalization of expression studies in the Eucalyptus genus.

SUBMITTER: de Oliveira LA

PROVIDER: S-EPMC7107212 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Reference genes for the normalization of gene expression in eucalyptus species.

de Oliveira Luisa Abruzzi LA Breton Michèle Claire MC Bastolla Fernanda Macedo FM Camargo Sandro da Silva Sda S Margis Rogério R Frazzon Jeverson J Pasquali Giancarlo G

Plant & cell physiology 20111224 2

Gene expression analysis is increasingly important in biological research, with reverse transcription-quantitative PCR (RT-qPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Considering the increased sensitivity, reproducibility and large dynamic range of this method, the requirements for proper internal reference gene(s) for relative expression normalization have become much more stringent. Given the increasing interest in the functional ...[more]

PMID: 22197885

Similar Datasets

Project description:BackgroundQuantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L).ResultsSpecific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups.qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59). LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and NormFinder-designated-reference genes.ConclusionsThe use of 2 different statistical algorithms results in the identification of different combinations of flax HKGs for expression data normalization. Despite such differences, the use of geNorm-designated- and NormFinder-designated-reference genes enabled us to accurately compare the expression levels of a flax MYB gene in different organs and tissues. Our identification and validation of suitable flax HKGs will facilitate future developmental transcriptomic studies in this economically-important plant.

Dataset Information

Reference genes for the normalization of gene expression in eucalyptus species.

Publications

Reference genes for the normalization of gene expression in eucalyptus species.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets