Abstract
To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta.
Highlights
The use of reference gene(s) as internal control for the measurement of target gene expression variation is the currently preferred method for normalizing real-time quantitative PCR (RT-qPCR) data because reference genes can capture all nonbiological variations [1]
All PCRs displayed efficiency between 93.5% and 104%, as shown in Transcript Profiles of Reference Genes The expression profiles of the five candidate reference genes were assessed in different developmental stages, castes and tissues (Figure 1a, 1b, 1c)
The Ct values for rpl18, ef1-beta, act and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were significantly different among different tissues (OneWay ANOVA, P#0.021), whereas no significant differences were detected in tbp expression in different tissues (Figure 1c)
Summary
The use of reference gene(s) as internal control for the measurement of target gene expression variation is the currently preferred method for normalizing real-time quantitative PCR (RT-qPCR) data because reference genes can capture all nonbiological variations [1]. Php?type = reference]) which can analyze the stability of gene expression. Several genes, such as ribosomal protein L18 (rpl18) [5,6,7], translation elongation factor 1 (ef1-beta) [8,9], actin (act) [8,9], glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [8,9] and TATA box binding protein (tbp) [10] have been used widely as candidate reference genes for gene expression studies in Insecta and other groups of organisms [11,12]. The expression of reference genes can differ widely in their stability over developmental stages [13,14,15,16], in different tissues [17,18,19,20,21] and under different environmental conditions [14,22,23]. A rational optimization approach to select and standardize reference genes is an important requirement in RT-qPCR-based transcriptome studies [24]
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