The estimation of bloodstain age utilizing circRNAs and mRNAs biomarkers.

Determining the time since deposition (TsD) of bloodstains can provide forensic investigators with additional clues, as it can corroborate eyewitness accounts, limit the number of suspects, and help confirm alibis. Bloodstains are the most common bodily fluid stains at crime scenes. In this study, we examined the relative expression levels (REs) of circRNAs and mRNAs data in bloodstains over ten time points by Real-time quantitative polymerase chain reaction (qPCR), to determine the utility of the relative expression levels of RNA markers for TsD estimation. Forensic samples more than just appear in indoor settings, we also evaluated the use of RNA degradation rate to indicate the age of bloodstains in different environments including indoor and outdoor conditions. The expression levels of six blood-specific mRNA markers (GYPA, CD93, ALAS2, SPTB, HBB, HBA), three highly expressed circRNAs in human peripheral blood (hsa_circ_0001445, hsa_circ_0000972, hsa_circ_0000095) and three reference genes (18 S, ACTB and U6) were analyzed across numerous ageing time points. Analysis of the degradation rates of individual RNAs under indoor and outdoor conditions showed that they exhibited a unique degradation profile during the four-month storage interval, with both circRNAs and mRNAs linearly showing continuous degradation, while U6 is more stable than other reference gene markers. In the current study, we firstly used circRNAs as additional novel biomarkers for bloodstain age estimation, and at the same time proved that different environments had a significant impact on the REs of certain blood biomarkers, and sex differences did not affect the age estimation of bloodstains. The REs of the selected RNA molecules in this study showed a non-linear relationship with bloodstain age and the mathematical formula for estimating the bloodstain age based on the relative expression levels of hsa_circ_0001445, ALAS2 and HBB can be used to estimate the TsD of bloodstains from the REs of bloodstains of unknown age, which represent a potentially effective approach to looking for time-dependent changes and TsD estimation.