Establishing Practical and Cost-Effective Methodologies for Preservation and Extraction of Environmental RNA (eRNA) From Aquatic Samples

Sampling environmental nucleic acids (eDNA and eRNA, collectively eNA) in aquatic systems facilitates the efficient exploration of the distribution of aquatic species. Since eRNA is inherently less stable than eDNA, eRNA may yield more spatiotemporally precise information about organisms, but this volatility also poses unique methodological challenges, making it unclear whether eDNA protocols are suitable for eRNA samples. To address this gap, we conducted experiments using naturally occurring eRNA and eDNA from the mitochondrial cytochrome b (CytB) gene from largemouth bass (Micropterus salmoides) along with DNA and RNA extracted from bluegill tissue (Lepomis megalotis). We first conducted a degradation experiment to test the efficacy of refrigeration for eRNA preservation (specifically mitochondrial messenger eRNA, emRNA), given its widespread use for eDNA samples. We then compared a suite of approaches for the long-term preservation and co-extraction of emRNA and eDNA in the laboratory. Despite the potential that the instability of eRNA might render eDNA approaches unsuitable, we found that methods developed for eDNA samples can be readily repurposed for emRNA. Contrary to our hypothesis that refrigeration of water samples might not efficiently preserve emRNA in the short term, samples in a cooler on ice showed no decrease in concentration or detectability over 25 h for largemouth bass (Tukey's, all p > 0.3). Filtration time (i.e., time spent at room temperature) also slightly reduced eRNA yields, with concentration decreasing as filtration time increased for largemouth bass emRNA (LM, p = 0.05), highlighting the importance of cold conditions throughout processing. Additionally, as we hypothesized, a low-cost chloroform extraction protocol yielded equivalent emRNA quantities to more expensive commercial kits for both largemouth bass and bluegill (Tukey's, p between methods > 0.8), in some cases even yielding higher concentrations than these methods. Overall, these results demonstrate the possible practical feasibility of eRNA sampling and provide guidance for future work on eRNA in natural systems.