Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses

Authors: OSBORNE, CAMERON - Kansas State University; COOPER, ANASTASIA - Kansas State University; HALL, BRANDON - Kansas State University; BIRD, EDWARD - Kansas State University; Nayduch, Dana; SILVER, KRISTOPHER - Kansas State University

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/27/2023
Publication Date: 10/4/2023
Citation: Osborne, C., Cooper, A., Hall, B., Bird, E., Nayduch, D., Silver, K. 2023. Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses. Scientific Reports. 13:16729. https://doi.org/10.1038/s41598-023-43750-2.
DOI: https://doi.org/10.1038/s41598-023-43750-2

Interpretive Summary: Differential gene expression is a scientific approach where the researcher determines whether target genes of interest are producing more or less products in order to understand the role of those genes in biological processes of interest. Critical to this approach is having a reference gene that serves as a veritable common denominator which allows for comparisons of gene expression across different tissues, treatments or conditions such as experimental treatments vs. controls, or different life stages, etc. In this study, candidate reference genes were tested for their utility in gene expression analyses in the biting midge Culicoides sonorensis. Six candidate reference genes were evaluated under seven conditions, treatments and tissues using four analysis tools, and scores were ranked for each. Ultimately, the findings showed that optimal reference genes varied between conditions and that just two reference genes were necessary for each condition. The candidate reference genes elucidated in this study can be used in future experiments aimed at understanding the biology, ecology and behavior of this important agricultural pest, which will ultimately help scientists uncover targets for their management and control.

Technical Abstract: Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require using a set of stably expressed reference genes for normalization to produce accurate results. The biting midge Culicoides sonorensis is an important vector of livestock pathogens in the United States and is often used as a model species for related biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, ß-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, a six developmental midge stages, four adult female body parts and tissue groups, and females injected with bluetongue virus (BTV) or vesicular stomatitis virus (VSV). Stable gene expression was assessed using four analysis tools (RefFinder, NormFinder, geNorm, and BestKeeper). The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. Ultimately, the findings show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis.