Evaluating Plasma-Handling Effects on Extracellular Vesicle RNA Yield and Cargo Profiles in Fish: A Methodological Insight.

Extracellular vesicle (EV)-derived RNA (EV-RNA) is emerging as a valuable biomarker source in aquatic species. However, the composition and stability of long RNA species, particularly messenger RNA (mRNAs) and long non-coding RNAs (lncRNAs), remain largely unexplored in teleosts. Pre-analytical factors such as input plasma volume and storage conditions may influence RNA recovery, especially in low-input samples like fish plasma. Here, we evaluated the effects of input sample volume and freezing storage on EV-RNA yield and integrity in gilthead sea bream (Sparus aurata) plasma using the exoRNeasy commercial kit. Both Maxi (∼2 mL) and Midi (∼1 mL) kit formats produced comparable RNA recovery, indicating flexibility in plasma volume requirements. Freezing did not alter the overall RNA biotype composition or the identity of the dominant transcripts but resulted in a higher proportion of shorter and incomplete isoforms, suggesting selective susceptibility to degradation of long RNAs during freezing storage. Using Oxford Nanopore Technologies long-read sequencing, we generated the first long RNA-resolved EV transcriptome for this species. EV cargo was dominated by ribosomal and metabolic mRNAs, consistent with conserved vertebrate EV signatures, and 302 distinct lncRNAs were identified, two of which accounted for nearly 90% of lncRNA reads. Predicted interactions between abundant lncRNAs, EV mRNAs, and their encoded ribonucleoproteins suggest coordinated packaging of RNA-protein modules. Overall, this study validates exoRNeasy for fish plasma and provides the first comprehensive long-RNA EV landscape in a teleost, providing a valuable framework for future molecular and functional studies in non-model fish species.