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.

OBJECTIVE

To determine how microRNA (miRNA) levels are altered in serum samples under different storage conditions.

METHODS

Whole blood was collected from 10 healthy dogs and placed into 7 tubes: (Tube 1) a red-top tube (RTT) centrifuged for immediate serum separation and stored at -80 °C (control), (Tube 2) room temperature (RT) for 24 to 36 hours, or (Tube 3) refrigerated temperature (FT) for 24 to 36 hours; (Tube 4) an RTT stored at RT for 24 to 36 hours or (Tube 5) FT for 24 to 36 hours; and (Tube 6) a serum separator tube centrifuged and stored at RT for 24 to 36 hours or (Tube 7) FT for 24 to 36 hours. Following RNA isolation and sequencing, differential expression analysis was performed.

RESULTS

All tubes had altered levels of miRNA compared to the control. Tube 5 was not further analyzed due to a high incidence of hemolysis. Conditions with immediate serum separation and refrigeration (Tube 3, Tube 7) had the fewest alterations in miRNA levels.

CONCLUSIONS

Collection of serum for future miRNA analysis should be done with immediate separation of serum (via centrifugation of an RTT and removal of serum or use of a serum separator tube) and storage at FT for 24 to 36 hours prior to placement into a -80 °C freezer to have the fewest changes in miRNA levels.

CLINICAL RELEVANCE: These findings will help facilitate collection of additional samples from secondary research sites. Researchers should be aware of specific miRNA that may be altered based on storage conditions.