RNA degradation of blood reveals a time diagnostic window in a controlled environment

Determining the age, or time since deposition (TSD), of bloodstains would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. The physicochemical changes that occur to major biomolecules as a bloodstain degrades can be used to approximate the TSD of bloodstains. Our study aims to quantify the timewise and temperature degradation trends found in total RNA from bloodstains, expanding the scope of the TSD research which has previously explored DNA and targeted mRNA molecules. Whole bovine blood was stored in plastic microcentrifuge tubes at 21˚C or 4˚C and tested over different timepoints spanning one week. A total of nine RNA metrics were visually assessed and quantified using linear and mixed models. The RNA Integrity Number equivalent (RINe) and the DV200 demonstrated strong relationships with time and independence from one another. Both exhibited negative trends over time, showing a decrease in both the quality and quantity of larger RNA fragments that varied with temperature. The RINe model fit was high (R2 = 0.60), and while including the biological replicate as a random effect increased the fit for all RNA metrics, no significant differences were found between biological replicates stored at the same temperature for the RINe and DV200 metrics; importantly, this suggests that both these standardized metrics can likely be directly compared between scenarios and individuals. The presence of a significant decrease in the retained RNA quality metrics after 24 hours suggests that this method could be used to differentiate day-old bloodstains from older bloodstains. Our models also demonstrated a faster rate of RNA degradation at room temperature than at 4˚C, supporting the need to develop regional models. This study provides a novel approach for blood TSD estimates, producing metrics that are not affected by inter-individual variation and improving our understanding of the degradation occurring in bloodstains, bringing us one step closer to eventual crime scene application.