Multi-Species Tardigrade RNA Isolation Protocol.

Tardigrades are microscopic animals that are known for their exceptional tolerance to environmental stress. These animals can survive desiccation, extreme pressure, low temperatures, and high levels of ionizing radiation. However, the molecular mechanisms underlying these extremotolerant capabilities remain incompletely understood. The molecular approaches used to investigate these mechanisms, which include RNA sequencing, RNA interference (RNAi), and heterologous expression of tardigrade genes in systems such as bacteria, require the isolation of high-quality messenger RNA (mRNA) from limited biological material. In this study, we describe a reproducible protocol for isolating total RNA from tardigrades that is (1) effective across multiple species, including a heterotardigrade species, (2) requires only 25-200 animals, depending on body size, and (3) yields RNA of sufficient quantity and quality for downstream applications. The present protocol successfully enables efficient RNA recovery from reasonable numbers of tardigrades and supports molecular investigations of stress tolerance across various tardigrade species.