Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill

Abstract

BackgroundEucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings.ResultsBy the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs indentified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm.ConclusionOur study showed that expression stability varied between putative reference genes tested in E. globulus. Based on the AGO1 relative expression profile obtained using the genes suggested by the algorithms, H2B and TUA were considered as the most suitable reference genes for expression studies in E. globulus adventitious rooting. UBI and 18S were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation.

Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions

“…Here, ELF1B and ACTB, followed by TUA, were ranked as the most stable reference genes according to our GeNorm and NormFinder analyses, whereas the 18S gene was found to be the most unstable. Greater expression stability has already been demonstrated for the plant genes ELF1B (Jian et al, 2008;Hu et al, 2009), ACTB (Stolf-Moreira et al, 2011), and TUA (Jian et al, 2008;de Almeida et al, 2010). Previous plant gene expression studies have classified the 18S gene among the most stable (Stolf-Moreira et al, 2011;Xu et al, 2011), whereas in other studies, it is ranked as the least stable (de Almeida et al, 2010), which is consistent with our findings.…”

“…Compared with other studies of expression stability, such as adventitious rooting in Eucalyptus globulus (de Almeida et al, 2010) and Populus (Xu et al, 2011), in the different soybean tissues (Jian et al, 2008;Hu et al, 2009) and drought-stressed soybean roots and leaves (Stolf-Moreira et al, 2011), 18S transcripts were the most abundant (de Almeida et al, 2010;Stolf-Moreira et al, 2011). TUA2 [called TUB by Jian et al (2008)] and ELF1B exhibited the largest (Jian et al, 2008) and smallest (Hu et al, 2009) variations in Ct values, respectively.…”

Selection of optimized candidate reference genes for qRT-PCR normalization in rice (Oryza sativa L.) during Magnaporthe oryzae infection and drought

“…The most consistent result of the consensus rankings was that under all of the conditions, 18S rRNA ranked at or near the bottom. The gene 18S rRNA has been widely used as a reference gene for gene expression analyses for a number of species (Teste et al, 2009;de Almeida et al, 2010). However, herein, the data demonstrated that 18S displayed the lowest expression stability, with the lowest average gene ranking across all of the samples analyzed.…”

Reference gene selection for qPCR in Ammopiptanthus mongolicus under abiotic stresses and expression analysis of seven ROS-scavenging enzyme genes

“…2; Table 2) were ranked poorly based on the two softwares in this study, indicating that these genes should be avoided as internal controls when analyzing gene expression levels in A. mongolicus under abiotic stresses, whereas tubulin (Cortleven et al 2009;de Almeida et al 2010), ATPbinding cassettes transporter gene (Libauh et al 2008) and elongation factor gene (Nicot et al 2005;Tong et al 2009), have been reported to be invariant genes in other studies. Fig.…”