Gene Expression Profile Analysis is Directly Affected by the Selected Reference Gene: The Case of Leaf-Cutting Atta Sexdens

Abstract

Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes.

DoublesexEvolution Is Correlated with Social Complexity in Ants

“…2013; Helmkampf et al. 2015; Livramento et al. 2018), were used to normalize gene expression across different samples.…”

Validation and identification of reference genes in Chinese hamster ovary cells for Fc-fusion protein production

“…It belongs to the S9P family of ribosomal proteins 55 and showed stable gene expression in certain insects. 56 It is recommended by geNorm program that normalization with the combination of different genes is preferable for RT-qPCR analysis. 50 The involvement in various biological metabolism pathways would more likely to avoid gene co-regulation when used as reference.…”

Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes

“…For this, RefFinder (East Carolina University, Greenville, NC, USA) software is the most commonly used reference gene selection tool and is based on four other algorithms: geNorm (Vandesompele et al, 2002), NormFinder (Andersen et al, 2004 ), BestKeeper (Pfaffl et al, 2010) and Comparative ΔCt (Silver et al, 2006), forming a final ranking. This yields more appropriate and consistent results for the RT-qPCR techniques (Livramento et al, 2018). The RT-qPCR technique is a fast and costeffective method for assaying specific gene expression compared to other methods, such as whole transcriptome sequencing and is, thus, widely use in the cardiac field.…”

“…In this context, there is an enzyme named chymase, that may be involved in this heart condition, considering that it participates in 80% of cardiac angiotensin II synthesis (Ahmad et al, 2014) Research, Development, v. 9, n. 11, e1599119702, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9702 The Rattus norvegicus albinus specie is one of the most widely used animal models in experiments with metabolic alterations (Hunt et al, 2017), that can cause serious heart problems, and it is important to assess the timing of these changes. The alteration of the gene expression of certain genes may precede the histological or clinical changes in these animals (Khalilpourfarshbafi et al, 2017) and to verificate this event, it is necessary to evaluate the expression of the genes, which can be done by quantifying the messenger RNA (mRNA) of a given tissue, in a given time, and specific condition, through the real-time quantitative polymerase chain reaction (RT-qPCR) (Livramento et al, 2018).…”