A Comparative Protocol for Preserving Deep-Water Marine Invertebrate Tissues: DNA/RNA Shield vs. Liquid Nitrogen for Dual Extraction of High-Quality Nucleic Acids.

Preserving biological samples in the field is essential for ensuring high-quality nucleic acid extraction and reliable downstream molecular analyses. Broadly, two main preservation strategies are available: physical preservation, such as flash freezing in liquid nitrogen, which halts enzymatic activity by rapid cooling, and chemical preservation, using stabilizing reagents that inactivate nucleases and protect nucleic acids even at ambient temperatures. This protocol presents a comparative approach using liquid nitrogen and a commercial stabilizing reagent (DNA/RNA Shield, Zymo Research) to preserve tissue from five marine invertebrate species: two cold-water corals, two sponges, and one bivalve. Samples preserved by each method were processed with the AllPrep DNA/RNA Mini kit (Qiagen) to extract both RNA and DNA. RNA quality was assessed using RNA Integrity Number (RIN) scores. The stabilizing reagent preserved high-quality RNA in sponge and bivalve samples but did not prevent RNA degradation in coral tissues, which showed lower RIN scores compared to those preserved in liquid nitrogen. DNA yields were also consistently lower in tissues preserved with DNA/RNA Shield across all species. These findings suggest that DNA/RNA Shield can be a viable alternative to liquid nitrogen for some marine invertebrates, particularly in field conditions where cryopreservation is impractical. However, for cold-water corals, liquid nitrogen remains essential to ensure RNA integrity for transcriptomic analyses and other sensitive molecular applications (e.g., RT-qPCR). Key features • Comparative evaluation of preservation methods across five marine invertebrate species. • Liquid nitrogen is essential for maintaining high RNA integrity in cold-water corals, whereas DNA/RNA Shield effectively preserved RNA in sponges and bivalves. • Streamlined co-extraction protocol using the AllPrep DNA/RNA Mini Kit (Qiagen) enables reliable recovery of DNA and RNA from field-collected marine samples for downstream molecular applications. • Integrated quality control workflow (Qubit, Nanodrop, Bioanalyzer RIN) ensuring robust assessment of nucleic acid yield and integrity for downstream molecular applications.