Messenger RNA typing of environmental RNA (eRNA): A case study on zebrafish tank water with perspectives for the future development of eRNA analysis on aquatic vertebrates

• Published in: Environmental DNA (Hoboken, N.J.) Vol. 3; no. 1; pp. 14 - 21
• Main Authors: Tsuri, Kenji, Ikeda, Shizuya, Hirohara, Takaya, Shimada, Yasuhito, Minamoto, Toshifumi, Yamanaka, Hiroki
• Format: Journal Article
• Language: English
• Published: Hoboken John Wiley & Sons, Inc 01.01.2021; Wiley
• Subjects: Biodiversity; biological monitoring; Biomonitoring; Breeding; conservation of natural resources; Danio rerio; Deoxyribonucleic acid; DNA; Gene expression; Genes; Gills; Intestine; Keratin; Methods; Nonnative species; Physiology; Polymerase chain reaction; Proteins; Reverse transcription; Ribonucleic acid; RNA; Skin; Taxonomy; Temporal resolution; Tissues; Typing; Vertebrates; Water analysis; Water sampling; Wildlife; Zebrafish; Japan
• DOI: 10.1002/edn3.169
• ISSN: 2637-4943

As an alternative/supplement to conventional biodiversity survey methods, environmental DNA (eDNA) analysis has developed rapidly during the past decade, and is widely used for the biomonitoring of wildlife. However, the interpretation of eDNA results has been limited to the presence/absence or biomass/abundance of the target species because of uncertainty regarding the dynamics of eDNA in natural environments. This limitation may be ameliorated by targeting environmental RNA (eRNA). RNA is more prone to degrade than DNA and the pattern of messenger RNA (mRNA) expression changes depending on physiological conditions, meaning that the presence or concentration of mRNA could reflect the organism's presence with higher temporal resolution and provide information beyond simple presence/absence. Technical developments in the detection of eRNA focusing on mRNA with these distinct features could permit the advanced usage of genetic materials in water. In advancing this technique, we initiated this study asking that if we can detect elevated levels of eRNA whose genes are specific to a tissue source (e.g., gills or skin), then could not we infer the tissue origin of the genetic material detected. To this end, we developed gene‐specific primer sets for the target genes with biased expression in the gills, skin, and intestine, and conducted reverse transcription–polymerase chain reaction on zebrafish breeding tank water samples, obtaining positive results for all assays. The result of our experiment confirmed that the specific target tissues can be the source of genetic materials detected in water and with that we offer a proof of concept for eRNA analysis targeting specific mRNAs of aquatic vertebrates. In this commentary, we provide information on the experimental steps used for mRNA typing of eRNA from zebrafish as well as the limitations and challenges of this technique and the prospect of mRNA typing of eRNA in the future. We developed gene‐specific primer sets for the target genes with biased expression in the gills, skin, and intestine, and conducted reverse transcription‐polymerase chain reaction on zebrafish breeding tank water samples, obtaining positive results for all assays. This commentary provides information on the experimental steps used for mRNA‐typing of eRNA from zebrafish as well as the limitations and challenges of this technique and the prospect of mRNA‐typing of eRNA in the future.