Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae)

Abstract

The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is the main insect pest of peppers (Capsicum spp.) throughout the southern U.S. and a potential target for novel control methods that may require gene expression analyses. careful selection of adequate reference genes to normalize Rt-qpcR data is an important prerequisite for gene expression studies since the expression stability of reference genes can be affected by the experimental conditions leading to biased or erroneous results. the lack of studies on validation of reference genes for Rt-qpcR analysis in A. eugenii limits the investigation of gene expression, therefore it is needed a systematic selection of suitable reference genes for data normalization. In the present study, three programs (BestKeeper, geNorm and normfinder) were used to analyze the expression stability of candidate reference genes (β-ACT, ArgK, EF1-α, GAPDH, RPL12, RPS23, α-TUB, 18S and 28S) in A. eugenii under different experimental conditions. our results revealed that the most stably expressed reference genes in A. eugenii varied according to the experimental condition evaluated: developmental stages (EF1-α, 18S and RPL12), sex (RPS23 and RPL12), low temperature (GAPDH and α-TUB), high temperature (α-TUB and RPS23), all temperatures (α-TUB and GAPDH), starvation (RPL12 and α-TUB), and dsRNA exposure (α-TUB and RPL12). Our study provides for the first time valuable information on appropriate reference genes that can be used in the analysis of gene expression by Rt-qpcR in biological experiments involving A. eugenii.Reverse-transcription quantitative PCR (RT-qPCR) is widely used in gene expression studies due to its simplicity, reproducibility, high sensitivity, accuracy and cost-effectiveness 1,2 . Although RT-qPCR is considered a highly accurate technique, several experimental factors can lead to results that are not reliable measurements of gene expression. These factors include purity and integrity of RNA, quantity of starting RNA and cDNA, reverse transcription and PCR efficiency, and pipetting errors 3 . Thus, in RT-qPCR analysis is necessary to use reference genes to normalize the data in order to eliminate or at least reduce the technical variation among the tested samples and precisely estimate the expression of the target genes 4 .Usually, housekeeping genes related to basic cellular functions are used as reference genes in the normalization strategy because these genes are supposed to have constitutive and stable expression under a variety of physiological conditions and experimental treatments. However, several studies have demonstrated that the expression of housekeeping genes is not always stable and can be influenced by developmental stage, tissue, sex, and biotic or abiotic stresses that the organism is subjected 5-9 . Therefore, the selection of suitable reference genes according to the specific experimental conditions is essential to ensure accurate results.The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is the most economica...

Selection and Evaluation of Reference Genes for qRT-PCR in Spodoptera frugiperda (Lepidoptera: Noctuidae)

“…Previous research also indicates that ribosomal protein genes, as commonly used reference genes, have a wide range of applications in many insects. For example, RPL27 and RPL32 in different developmental stages and tissues of Apolygus lucorum (Meyer-Dur) [33], RPL12 in starvation treatment of Anthonomus eugenii (Cano) [9], and RPS20 in temperature treatment of Diaphorina citri (Kuwayama) [40]. However, the stability of ribosomal protein genes is not universal in all insects [24,34].…”

“…Quantitative real-time PCR (qRT-PCR) is an important molecular technology with high sensitivity, reliability, and specificity. It is always used in gene quantitative expression analysis and transcriptome verification [9][10][11][12]. However, many factors can lead to systematic errors and affect the accuracy of qRT-PCR results, such as the integrity and purity of total RNA, the efficiency of reverse transcription and PCR program, and pipetting [13][14][15].…”

“…Therefore, it is essential to use reference genes as the benchmark to eliminate various errors and normalize the qRT-PCR analysis [16]. Several traditional reference genes are always used as the benchmark to evaluate the expression profile of function genes, such as 18S ribosomal RNA (18S), alpha-tubulin (α-TUB), beta-actin (β-ACT), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) [9,10,17,18]. These reference genes are involved in the metabolism process of insect cells.…”

Selection and Validation of Reference Genes For qRT-PCR Analysis of Rhopalosiphum padi (Hemiptera: Aphididae)

“…Additionally, GAPDH activity is vital for membrane development, microtubule processing, and reactions catalyzed by phosphotransferases and kinases (Colell et al, 2009). Several recent studies have used GAPDH as a reference control for gene expression analyses (Han et al, 2020;Pinheiro and Siegfried, 2020;Yin et al, 2020). In Lucilia cuprina, GAPDH expression is highly variable, making the gene a poor choice as a reference control (Bagnall and Kotze, 2010).…”

“…Many factors considerably influence the threshold cycle (Ct) values, including RNA quality and quantity, variable transcriptional efficiencies, primer characteristics, and PCR conditions (Udvardi et al, 2008;Bustin et al, 2009). Consequently, identifying appropriate and reliable reference genes to serve as internal controls is essential for normalizing expression levels (Bustin, 2002;Pinheiro and Siegfried, 2020). In most analytical methods, the use of reference genes can eliminate the differences in sample purity and concentration, thereby enabling comparisons of target gene expression between samples (Radonić et al, 2004).…”