Validation of reference genes for quantitative real-time PCR in tiger beetles across sexes, body parts, sexual maturity and immune challenge

Abstract

Reference genes are frequently used as normalizers for expression studies despite not being previously verified to present suitable stabilities. Considering the interest that tiger beetles have generated in the past years, resulting in a variety of studies, it is crucial to dispose of a validated reference gene panel for expression studies. Nine candidate genes were tested in Cicindela campestris and Calomera littoralis across several conditions and their transcription levels were assessed with geNorm, NormFinder, BestKeeper and ΔCT method algorithms. Results showed high stabilities across sexes, immune challenge and gonad developmental stages for all genes tested, while body parts comparison presented less constant expression values. Only two genes are sufficient to perform a proper normalization for most of the conditions tested, except for the body parts comparison in C. littoralis, which requires the use of at least three reference genes. On the whole, no universal gene is found to be suitable for all situations, but according to the acceptable range of values, NADH, B-t, Vatpase and ArgKin seem to present the most constant expression stability, indicating their suitability as reference genes in most of the conditions. This is the first report evaluating the stability of housekeeping genes in adephagan beetles.

Reference Gene Selection for Normalizing Gene Expression in Ips Sexdentatus (Coleoptera: Curculionidae: Scolytinae) Under Different Experimental Conditions

“…Hence, this is the first report for suitable reference genes in any Ips bark beetles. Accurate normalization using a stable reference gene is necessary to conduct gene expression studies under specific experimental conditions and to avoid erroneous differences in target gene expression (Andersen et al, 2004;Bustin et al, 2005;Ferguson et al, 2010;Cheng et al, 2013;García-Reina et al, 2018;Xie et al, 2021). The results obtained in this study indicate that the stability of reference genes in ISx can differ under various experimental conditions, including developmental stage, sex, and tissue-specific conditions, and exposure to abiotic conditions (Tables 2-5 and Figures 1B-4) as observed in other reference gene finding studies in insects (Lu et al, 2018).…”

“…Vacuolar-type ATPase ( V-ATPase-A ) is a proton translocating pump responsible for ATP hydrolysis, one of the most highly conserved eukaryotic proteins. The V-ATPase-A gene had been commonly used as a reference gene in Amrasca biguttula for experiments involving starvation stress and different life stages (Singh et al, 2018 ); for sex-specific experiments in Cicindela campestris (García-Reina et al, 2018 ). On the contrary, V-ATPase-A gene expression was highly unstable in the developmental and tissue stages of Mythimna separata (Li et al, 2018c ).…”

“…Therefore, internal control genes (alternatively called reference genes) are commonly used to normalize mRNA levels for more accurate gene expression quantification as their expression should not vary under different biological or experimental conditions (Nicot et al, 2005 ; Lu et al, 2018 ). Furthermore, it is strongly recommended to use multiple reference genes for target gene expression normalization for more authenticity (García-Reina et al, 2018 ; Shakeel et al, 2018 ; Wang et al, 2020 ). There are several studies on gene function analysis in insects using reference genes such as β-actin ( Actin ), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), elongation factor 1α ( EF1A ), β-Tubulin (β -Tubulin ), ribosomal proteins ( RPs ), ubiquitin ( UbiQ ), superoxide dismutase ( SOD ), heat shock protein 90 ( HSP90 ), and vacuolar-type H+-ATPase subunit B ( V-ATPase-B ) for gene expression normalization (Lu et al, 2018 ; Qu et al, 2018 ; Gao et al, 2020 ; Wang et al, 2020 ).…”

Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

“…Since its publication in 2018, this article by Shakeel and colleagues on insects has been cited 22 times by articles in PubMed. Fourteen of these citing articles are those evaluating candidate reference genes [ 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 , 179 ]. We hope that our critical review will similarly stimulate future research on experimentally validating reference genes for gene expression studies in Aspergillus (and in fungi in general) using RT-qPCR.…”

Identifying optimal reference genes for gene expression studies in Eurasian spruce bark beetle, Ips typographus (Coleoptera: Curculionidae: Scolytinae)

“…It is pretty clear now that a single reference gene is not appropriate for the more comprehensive experimental conditions, and it can generate an error in the gene expression estimations causing nonoptimal interpretation of the data 25 , 34 , 35 . For mitigation, it is often recommended to use multiple reference genes to minimize variations by RT-qPCR normalization 23 , 36 , 37 . Several research studies revealed that most reference gene expression depends on samples/experimental conditions, suggesting no universal reference gene is available for all experimental conditions 38 .…”