Validation of Reference Genes in Solenopsis invicta in Different Developmental Stages, Castes and Tissues

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.

Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR

“…These genes have been widely used as internal standards for the normalization of RT-PCR and RT-qPCR data. During the last decade, many studies have revealed the instability of housekeep- ing gene expression (31,34,35), although they can also be acceptable internal standards under certain conditions. This fact is evidence that gene expression stability varies with the gene, species, tissue, and culture conditions used (36,37).…”

Selection and evaluation of potential reference genes for gene expression analysis in Avena fatua

“…Recent studies have shown that ACT expression can change in response to a variety of conditions (Ruan & Lai 2007;Zarivi et al 2015;Wrzesińska et al 2016). Thus, these traditionally used reference genes are not persistently and stably expressed in many species or different experimental treatments (Chandna 2012;Cheng et al 2013;Ma et al 2016), which emphasised the need to evaluate reference genes in A. fatua.…”

Validation of reference genes in leaf-cutting ant Atta sexdens rubropilosa in different developmental stages and tissues

“…Incorrect results might be obtained due to misinterpretation of RNA transcription levels especially for low abundance gene transcripts [10,11]. To date, several works were developed to determine the reference gene in Insecta, which demonstrate that is impossible to find a universal reference gene able to covering all organism and conditions [11,12,13,14,15,16,17]. The choice of a gene as reference gene is not trivial and starts with the selection of candidate reference genes to be analyzed [18].…”

“…The most studied reference genes, GAPDH and 18s rRNA, are not always expressed in a constant manner. In addition, their expression can be altered depending on the organisms and their life stages [9,12]. A good strategy for selecting potential candidate reference genes is based on previous data from species relative to the studied specie due to the high degree of similarity between genomes and the expectation of a similar expression level [9].…”

“…A good strategy for selecting potential candidate reference genes is based on previous data from species relative to the studied specie due to the high degree of similarity between genomes and the expectation of a similar expression level [9]. However, differences in stability have been verified in the analysis of a reference gene in Insecta for organisms from the same order [11,12,14,15], family [20,21] and even for those of the same genus [14,16,21]. This justifies studies to validate reference genes for an organism and experimental conditions before the analysis for precise mRNA quantification [22].…”