Validating post-enrichment steps in environmental RNA analysis for improving its availability from water samples

Environmental RNA (eRNA) analysis is expected to inclusively provide the physiological information of a population and community without individual sampling, having the potential for the improved monitoring of biodiversity and ecosystem function. Protocol development for maximizing eRNA availability is crucial to interpret its detection and quantification results with high accuracy and reliability, but the methodological validation and improvement of eRNA collection and processing methods are scarce. In this study, the technical steps after eRNA extraction, including genomic DNA (gDNA) removal and reverse transcription, were focused on and their performances were compared by zebrafish (Danio rerio) aquarium experiments. Additionally, this study also focused on the eRNA quantification variabilities between replicates and compared them between the PCR and sample levels. Results showed that (i) there was a trade-off between gDNA removal approaches and eRNA yields and an excess gDNA removal could lead to the false-negative eRNA detection, (ii) the use of the gene-specific primers for reverse transcription could increase the eRNA yields for multiple mitochondrial and nuclear genes compared with the random hexamer primers, and (iii) the coefficient of variation (CV) values of eRNA quantifications between PCR replicates were substantially lower for those between samples. Including the study, further knowledge for the sensitive and precise detection of macro-organismal eRNA should be needed for increasing the reliability and robustness of eRNA-based biomonitoring.