Microsatellite loci in urine supernatant and stored samples from racehorses.

This study investigates the possibility of extracting amplifiable DNA from the urine of racehorses, alongside factors in storage and transportation which may influence genotyping. The research revealed that DNA retrieval is feasible from both blood and urine samples, which success hinging on appropriate collection and storage techniques.

Research Methods

• Researchers collected and analyzed samples from 261 Thoroughbreds and Standardbreds horses. This included 580 urine samples, 279 whole blood samples, and 40 plasma samples
• They isolated genomic DNA from these stored samples and then evaluated and quantified this DNA.
• Five equine microsatellite loci (VHL20, HTG4, AHT4, HMS6, and HMS7) were selected and amplified using PCR techniques.
• The fragment sizes of the amplified loci were determined using capillary electrophoresis.

Results

• The study found that high-molecular-weight and amplifiable DNA could be retrieved successfully from refrigerated blood samples, while results for urine were variable.
• They found DNA in both urine supernatant (the clear liquid lying above a solid residue) and sediment.
• Subjecting urine samples to repeated freeze-thaw cycles led to an accumulation of amplifiable DNA in the supernatant, as well as clearance of naked DNA. However, multiple freeze-thaw cycles lowered DNA yield and caused DNA degradation, leading to a failure in detecting the microsatellite loci.
• The presence of certain drugs in tested samples did not hinder PCR amplification. However, contaminants in the DNA samples did interfere with PCR amplification and resulted in partial microsatellite profiles.

Conclusion

• By keeping and storing urine and blood samples properly, they can be successfully genotyped. The freeze-thaw cycles were found to be detrimental to the integrity of DNA within urine.
• Increases in the volume of urine used in the experiments resulted in a stronger recovery of DNA.