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.

Evaluation of reference genes for RT-qPCR studies in the leaves of rice seedlings under salt stress

“…Several genes are commonly known to be constitutive, and are regularly used as reference genes in RT-qPCR analyses (Wang et al, 2012). However, even genes involved in basic cellular metabolism processes can exhibit significant variations in expression that make them unsuitable as internal controls in certain situations (Czechowski et al, 2005;Gutierrez et al, 2008;Guénin et al, 2009;de Oliveira et al, 2012). The ideal reference gene should have the same expression, regardless of the experimental conditions.…”

“…Without this standardization, incorrect results may be obtained (Jain et al, 2006). These constitutive genes are termed reference, normalizer, or standard genes in the RT-qPCR technique (de Oliveira et al, 2012).…”

“…However, the constitutive gene should not exhibit significant variation in expression because it is a reference gene, but the target may have different expression patterns within the experiment (Expósito-Rodríguez et al, 2008). The genes chosen as references are usually involved in basic cellular processes such as cell structure maintenance and primary metabolism (Czechowski et al, 2005;de Oliveira et al, 2012). Although several genes are routinely used and reported as good reference genes in studies with plants, the genes encoding 18S rRNA (18S rRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF-1α), actin (ACT), and α tubulin (α-TUB) (Benitez et al, 2013) have shown differential expression between tissues, genotypes, and ambient test conditions (Czechowski et al, 2005;Expósito-Rodríguez et al, 2008;Gutierrez et al, 2008).…”

Identification and Validation of Reference Genes for Transcript Normalization in Strawberry (Fragaria × ananassa) Defense Responses

“…For this reason we strongly recommend to perform a validation of the putative reference genes prior any quantitative expression studies, as recommended elsewhere [50], [23], [51], [52], [19].…”

The Eucalyptus Tonoplast Intrinsic Protein (TIP) Gene Subfamily: Genomic Organization, Structural Features, and Expression Profiles

“…Each reaction was performed in triplicate in a total volume of 10 μl, and contained 20 ng of cDNA and 0.2 μM of each EgTIP -specific primer ( Supplementary Table S1 ). Among five selected E. grandis reference genes ( Actin , GAPDH , Cdk8 , Transcription elongation factor s-II , and Aspartyl-tRNA synthetase ; de Oliveira et al, 2012) tested, actin was the most stable and thus employed as endogenous control ( Supplementary Table S1 ; Goicoechea et al, 2005). Cycle threshold (Ct) values were obtained for each sample, and relative quantification was determined using the 2 -ΔΔCt method as described (Livak and Schmittgen, 2001) Amplification efficiencies were derived from the amplification plots using the program LinRegPCR (Ramakers et al, 2003).…”