Abstract
Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying gene expression across various biological processes, of which requires a set of suited reference genes to normalize the expression data. Coleomegilla maculata (Coleoptera: Coccinellidae), is one of the most extensively used biological control agents in the field to manage arthropod pest species. In this study, expression profiles of 16 housekeeping genes selected from C. maculata were cloned and investigated. The performance of these candidates as endogenous controls under specific experimental conditions was evaluated by dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt method. In addition, RefFinder, a comprehensive platform integrating all the above-mentioned algorithms, ranked the overall stability of these candidate genes. As a result, various sets of suitable reference genes were recommended specifically for experiments involving different tissues, developmental stages, sex, and C. maculate larvae treated with dietary double stranded RNA. This study represents the critical first step to establish a standardized RT-qPCR protocol for the functional genomics research in a ladybeetle C. maculate. Furthermore, it lays the foundation for conducting ecological risk assessment of RNAi-based gene silencing biotechnologies on non-target organisms; in this case, a key predatory biological control agent.
Highlights
One of the major ecological concerns regarding the RNAi-based gene silencing biotechnologies is their potential adverse impacts on non-target organisms (NTOs)[13,14,15,16,17]
Each candidate reference gene should be evaluated under specific experimental conditions[42,50]
Our results demonstrate that the suitable reference genes can be different in response to diverse biotic and abiotic conditions (Table 2; Fig. 3)
Summary
One of the major ecological concerns regarding the RNAi-based gene silencing biotechnologies is their potential adverse impacts on non-target organisms (NTOs)[13,14,15,16,17]. The normalization bias caused by a single, non-validated reference gene has been shown to lead to unreliable results and questionable conclusions, especially with tissue samples[33,40] To counter this bias, using two to five validated stably expressed reference genes is the most appropriate approach to normalize RT-qPCR data[41]. Despite the demonstrated necessity for systematic selection and validation of reference genes in RT-qPCR studies[42], insufficient normalization, especially, relying on non-validated (single) reference genes is still a common practice[38,39] This is of particular concern as the risks associated with RNAi-based gene silencing biotechnologies on NTOs could be subtle changes in gene expression. Without sufficient selection and validation, unreliable gene expression results can lead to erroneous risk assessments and risk decisions
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