Microsatellite loci in urine supernatant and stored samples from racehorses.

OBJECTIVE

To evaluate whether urine supernatant contains amplifiable DNA and to determine factors that influence genotyping of samples from racehorses after storage and transportation.

SAMPLE POPULATION

580 urine, 279 whole blood, and 40 plasma samples obtained from 261 Thoroughbreds and Standardbreds.

PROCEDURES

Genomic DNA was isolated from stored blood and urine samples collected from racehorses after competition. Quantified DNA was evaluated to determine whether 5 equine microsatellite loci (VHL20, HTG4, AHT4, HMS6, and HMS7) could be amplified by use of PCR techniques. Fragment size of each amplified locus was determined by use of capillary electrophoresis.

RESULTS

High-molecular-weight and amplifiable DNA were recovered from refrigerated blood samples, but recovery from urine varied. Deoxyribonucleic acid was recovered from both urine supernatant and sediment. Freeze-thaw cycles of urine caused accumulation of amplifiable DNA in the supernatant and clearance of naked DNA. Repeated freeze-thaw cycles significantly decreased DNA yield and induced DNA degradation, which resulted in failure to detect microsatellite loci. Select drugs detected in test samples did not affect PCR amplification. Contaminants in DNA isolates inhibited PCR amplification and resulted in partial microsatellite profiles.

CONCLUSIONS AND CLINICAL RELEVANCE

Properly stored urine and blood samples were successfully genotyped, but subjecting urine to freeze-thaw cycles was most detrimental to the integrity of DNA. Increasing the volume of urine used improved recovery of DNA.