Using total RNA quality metrics for time since deposition estimates in degrading bloodstains

The physicochemical changes occurring in biomolecules in degrading bloodstains can be used to approximate the time since deposition (TSD) of bloodstains. This would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. Our study aims to quantify the timewise degradation trends and temperature dependence found in total RNA from bloodstains without the use of amplification, expanding the scope of the RNA TSD research which has traditionally targeted mRNA and miRNA. Bovine blood with ACD-A anticoagulant was deposited and stored in plastic microcentrifuge tubes at 21 or 4°C and tested over different timepoints spanning 1 week. Total RNA was extracted from each sample and analyzed using automated high sensitivity gel electrophoresis. Nine RNA metrics were visually assessed and quantified using linear and mixed models. The RNA Integrity Number equivalent (RINe) and DV200 were not influenced by the addition of anticoagulant and demonstrated strong negative trends over time. The RINe model fit was high (R