Real-time RT-PCR quantitative assays and postmortem degradation profiles of erythropoietin, vascular endothelial growth factor and hypoxia-inducible factor 1 alpha mRNA transcripts in forensic autopsy materials

Abstract

Recent advances in molecular biology have suggested the potential usefulness of mRNA analyses in postmortem investigations of fatal mechanisms. The aim of the present study was to establish quantitative assays of oxygen-regulated factors including erythropoietin (EPO), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 alpha (HIF1A) mRNAs, and to investigate the postmortem stability of these mRNA transcripts in forensic autopsy materials. Relative quantification of EPO, VEGF and HIF1A mRNAs, based on the TaqMan reverse transcription-polymerase chain reaction (RT-PCR), was performed on autopsy tissue specimens from the heart ( n=10), brain ( n=10), kidney ( n=16) and lung ( n=8) after preservation at room temperature for various storage times. VEGF and HIF1A mRNA gradually degraded in patterns similar to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA used as an endogenous reference. Accordingly, the relative quantification of VEGF/GAPDH and HIF1A/GAPDH changed little up to 48 h postmortem in tissue samples from the brain, kidney and lung except for a mild deviation of HIF1A in the myocardium. However, the status was different for EPO mRNA, with extraordinary stability for postmortem degradation and a marked postmortem time-dependent increase in the EPO/GAPDH ratio for all tissue samples. The present study suggested the potential for applying quantitative analyses of mRNA transcripts to autopsy materials and indicated the significance of investigating degradation profiles prior to carrying out relative quantification of target mRNAs in autopsy materials.