Gene expression profiling for forensic age assessment of porcine skin wounds

Abstract

Determining the age of wounds is of utmost importance in both veterinary and human forensic pathology. The aim of this study was to design and optimize quantitative polymerase chain reaction (qPCR) primers for use on degraded RNA samples obtained in veterinary forensic cases, hereby ensuring assay robustness. Moreover, the aim was to evaluate if an expression signature, based on optimized short-amplicon primers, was able to differentiate porcine experimental granulation tissue according to age and if this could be used for wound age assessment in veterinary forensic cases. Initially, 12 samples of experimental granulation tissue (n=6) and skin (n=6) from two pigs were deliberately exposed to RNA degrading conditions before being stored in RNAlater. A panel of 24 robust primers were selected based on the intentionally degraded samples. Granulation tissue (5, 10, 15, 20, 25, 30, and 35 days of age) (n=94) and control skin (n=47) from 47 experimental pigs was sampled and stored in RNAlater. Furthermore, granulation tissue and fibrous scar tissue were sampled from 14 veterinary forensic cases. Microfluidic qPCR was performed to evaluate the gene expression of 24 genes. An expression signature of 14 genes reflected the age of the experimental wounds. The 5-day old wounds displayed the biggest divergence from the control skin. As the granulation tissue matured, gene expression gradually approached the levels observed in intact skin. The forensic samples clustered somewhat separately from the experimental samples. In conclusion, granulation tissue from the experimental wounds displayed a time-dependent expression profile based on 14 short-amplicon primers suitable for use with low-quality RNA. However, an expression profile of 14 genes cannot be used as the sole method for forensic age assessments of porcine wounds.