Selection and Evaluation of Reference Genes for Expression Analysis Using qRT-PCR in the Beet Armyworm Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

Abstract

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. The most common method for analyzing qRT-PCR data is to normalize mRNA levels of target genes to internal reference genes. Evaluating and selecting stable reference genes on a case-by-case basis is critical. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for normalization of mRNA expression in qRT-PCR analysis of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). For this purpose, three software tools (geNorm, NormFinder and BestKeeper) were used to investigate 10 candidate reference genes in nine developmental stages and five different tissues (epidermis, head, midgut, fat body and hemolymph) in three larval physiological stages (molting, feeding and wandering stages) of, S. exigua. With the exception of 18S ribosomal RNA (18S), all other candidate genes evaluated, β-actin1(ACT1), β-actin2 (ACT2), elongation factor1(EF1), elongation factor 2 (EF2), Glyceralde hyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), α-tubulin (TUB),proved to be acceptable reference genes. However, their suitability partly differed between physiological stages and different tissues. L10, EF2 and L17A ranked highest in all tissue sample sets. SOD, ACT2, GAPDH, EF1 and ACT1 were stably expressed in all developmental stage sample sets; ACT2, ACT1 and L10 for larvae sample sets; GAPDH, ACT1 and ACT2 for pupae and adults; SOD and L17A for males; and EF2 and SOD for females. The expression stability of genes varied in different conditions. The findings provided here demonstrated, with a few exceptions, the suitability of most of the 10 reference genes tested in tissues and life developmental stages. Overall, this study emphasizes the importance of validating reference genes for qRT-PCR analysis in S. exigua.

Selection of reference genes for qRT‐PCR and expression analysis of high‐altitude‐related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient

“…EF‐1 α was the best RG for grassland caterpillars from divergent altitudinal environments, as observed in other lepidopteran insects, such as Spodoptera litura under different developmental stages (Zhu et al., 2014), Plutella xylostell under diverse experimental conditions (developmental stages, tissues, and strains) (Fu et al., 2013), larval Sesamia inferens under insecticide exposure (Lu et al., 2015), and Plodia interpunctella under different strains (Tang et al., 2016). This reflects the stable expression of EF‐1 α in lepidopteran insects under many experimental conditions, and this gene should be considered as a candidate RG in the gene expression analysis of lepidopteran insects in the future.…”

Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.)

“…Although there have been extensive studies on selection and validation of reference genes in Lepidoptera insects Shakeel et al 2015;Sun et al 2015;Teng et al 2012;Zhu et al 2014), to the best of D r a f t our knowledge, two papers have been published regarding P. xylostella Teng et al 2012 glycerol-3-phosphate dehydrogenase-2 (G3PDH), E2F transcription factor 4 (E2F), and ribosomal protein 49 (rp49) Teng et al 2012). In this study, a comprehensive and systemic assessment of candidate reference genes from the P. and Delta Ct) and a web-based tool (RefFinder).…”

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

“…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].…”