Old meets new: Comparative examination of conventional and innovative RNA-based methods for body fluid identification of laundered seminal fluid stains after modular extraction of DNA and RNA.

The knowledge about the type of the body fluid/tissue that contributed to a trace can provide contextual insight into crime scene reconstruction and connect a suspect or a victim to a crime scene. Especially in sexual assault cases, it is important to verify the presence of spermatozoa. Victims often tend to clean their underwear/bedding after a sexual assault. If they later decide to report the crime to the police, in our experience, investigators usually do not send laundered items for DNA examination, since they believe that analysis after washing is no longer promising. As not only the individualization of traces on laundered items could be important in court, but also the type of biological material, we compared the potential of modular DNA and RNA extraction from the same specimen for simultaneous body fluid identification (BFI) and STR profiling of laundered items. BFI included the comparison of a broad range of conventional approaches, as wells as new molecular mRNA- and miRNA-based methods. The examination comprises the assessment of different fabrics and washing temperatures and multiple washing steps. Our results indicate that although conventional enzymatic and immunochromatographical approaches show limitations for BFI of laundered stains, the RSID test was sensitive enough to detect seminal fluid in 80% of all tested samples. Furthermore, the HY-Liter fluorescence analysis successfully detected spermatozoa, even in cloths that were washed twice. For the first time, it could be shown that a marker set of mRNAs can be applied for the identification of seminal fluid stained cotton and synthetic fiber fabrics that have been washed at 40 °C. Our experiments demonstrate that analysis of DNA and RNA of laundered items is feasible, giving the possibility to identify the perpetrator as well as the biological properties of the same laundered (seminal) stain under certain conditions.