Estimating time since deposition using quantification of RNA degradation in body fluid-specific markers

The first appearance of ribonucleic acid (RNA) in forensic science research was in 1984 in the study of post-mortem tissues. Since then, many studies have explored the role of gene expression and its potential applications in forensic science. The two main RNA molecules that have been subject to increasing interest in the forensic science community are messenger RNA (mRNA) and microRNA (miRNA). Identification of body fluid type and estimating the time since deposition can be of immense value to criminal investigations. Determining the time since deposition or age of a biological stain can help to indicate either when a crime happened, or whether the biological evidence was deposited before/after a known crime event, in order for samples to be excluded. The research presented here has used reverse transcription quantitative PCR to examine the relative expression ratio (RER) in two types of body fluid-specific markers (saliva and semen), to develop a method to estimate the age of biological stains. mRNA and miRNA markers specific to saliva and semen, along with three reference genes were selected. Biological samples from 20 participants were stored in a dark dry place at room temperature to simulate natural ageing. A series of desired ageing points were set and total RNA was extracted when samples reached each desired point. The degradation behaviour of each RNA marker was analysed, showing that they exhibited unique degradation profiles across a one-year storage interval for saliva and semen samples, where miRNAs and the U6 reference gene were shown to have high stability. The RERs exhibit a non-linear relationship with body fluid stain age and can be considered as a potential method for body fluid stain age estimation, hence the time since deposition.