Quantification of RNA degradation of blood-specific markers to indicate the age of bloodstains

Determining the time since deposition of a biological stain can provide essential information to a police investigation; indicating either when a crime occurred, or whether the biological evidence was deposited at the time of a known crime event. Bloodstains are one of the most important biological evidence types to forensic investigators. This research has used reverse transcription quantitative PCR to examine the relative expression ratio (RER) between different types of blood-specific markers, with the aim of developing a method to estimate the age of bloodstains. Targets included three mRNA markers (HBA, PBGD, HBB) and two microRNA markers (miR16, miR451), along with three reference genes (18S rRNA, ACTB mRNA, U6 snRNA). Blood samples from 10 individuals were deposited onto cotton swabs and stored at room temperature to simulate natural ageing. When samples reached a series of desired age points, total RNA was extracted. Analysis of the degradation rate of individual RNAs showed they exhibited unique degradation profiles during the nine-month storage interval, where miRNAs and U6 were shown to be more stable than other markers. The RERs show a non-linear relationship with bloodstain age, and were shown to be useful for bloodstain age estimation