Immediate separation and storage of serum at a refrigerated temperature for 24 to 36 hours leads to minimal alterations in circulating microRNA levels in dogs.

Immediate serum separation followed by refrigerated storage best preserves canine miRNA integrity for 24–36 hours.

This study investigated how storage conditions affect circulating microRNA (miRNA) levels in canine serum samples, with the goal of optimizing pre-analytical protocols for miRNA-based research. Whole blood from 10 healthy dogs was collected into red-top tubes (RTTs) or serum separator tubes (SSTs) and subjected to various storage conditions prior to RNA isolation and sequencing. Conditions included immediate serum separation followed by storage at room temperature (RT) or refrigerated temperature (FT) for 24–36 hours, as well as delayed separation with RT or FT storage. All conditions produced some degree of miRNA alteration compared to the control (immediate separation and storage at -80°C). Notably, RTTs stored without centrifugation at refrigerated temperature had a high rate of hemolysis and were excluded from further analysis. Samples with immediate serum separation—either via RTT centrifugation or SST—followed by refrigerated storage for 24–36 hours demonstrated the fewest changes in miRNA levels. The authors recommend this workflow for multi-site studies where immediate freezing at -80°C is not feasible. These findings are particularly relevant for veterinary oncology and biomarker research, where miRNA profiling is increasingly explored as a diagnostic and prognostic tool. Researchers should remain cognizant that certain miRNAs may still be differentially affected depending on storage conditions, and protocols should be standardized across collection sites to minimize pre-analytical variability.

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