Challenges in mRNA-based time since deposition estimation: A multiplex MPS primer panel investigation

Estimating the time since deposition (TsD) of biological stains can provide crucial information for crime scene investigations, but remains a challenging task. In this study, we investigated mRNA degradation patterns across four forensically relevant body fluids - blood, semen, menstrual blood, and vaginal secretion - using a custom-designed mRNA MPS primer panel. The primer panel included candidate mRNA markers identified by RNA-Seq for TsD estimation in multiple body fluids. It was designed to assess mRNA degradation patterns based on three aspects: (1) individual mRNA degradation trends, (2) intra-transcript stability, examining 5'- versus 3'-end degradation, and (3) inter-transcript stability, based on relative degradation between two targets. The results revealed high inter-individual variability, especially for low-abundance transcripts. Some mRNA markers showed degradation patterns that correlated with TsD. However, inconsistencies in the degradation patterns observed across the analyzed time series highlight the challenges associated with primer panel optimization, RNA extraction methods, and reproducibility across samples. Our results suggest that, while mRNA degradation remains a promising strategy for TsD estimation, its forensic application requires careful marker selection and validation across independent data sets, to account for methodological and biological variability.