Relative gene expression analysis of catalase in environmental RNA from Japanese medaka exposed to toxic chemicals

Environmental RNA (eRNA) has the potential as a non-invasive tool for assessing the physiological status of macro-organisms, yet the quantitative method for relative gene expression analysis is still underexplored. To bridge this gap, this study introduces a relative quantification method for eRNA, employing quantitative PCR (qPCR) to evaluate the expression of the antioxidant gene, catalase (cat), in the Japanese medaka Oryzias latipes exposed to two toxic chemicals for 96 h: chlorpyrifos (CPS) and carbamazepine. Our results showed that, in eRNA, genes frequently used as a reference for tissue or cells in conventional expression analysis correlated with each other. Also, one of them (elfa) exhibited less variability, showing its potential suitability as a reference gene in eRNA analyses. Additionally, cat expression levels in eRNA increased with increasing CPS concentrations, in a concentration-response manner. These results suggest the promising potential of qPCR applications for eRNA in the monitoring of an organism's health and response to environmental changes. However, we observed disparities in gene expression levels of cat and beta-actin (actb) between eRNA and whole fish body, indicating eRNA might have a biased origin. Further research is needed to uncover the origin of eRNA and determine the limitations and applicability domain of eRNA analysis.