Investigation of the potential for use of various mirna markers in blood identification

In the context of forensic science, identifying the source of body fluids left at a crime scene is crucial for reconstructing the events that took place. Traditional and literature-based methods for body fluid identification-particularly serology-based techniques-are often limited by factors such as sample consumption, labor intensity, time requirements, varying sensitivity levels, and lack of specificity. While DNA-based methods are prominent in personal identification, RNA-based approaches have gained importance in the identification of body fluid types, which is a key parameter for crime scene reconstruction. Literature reviews indicate that microRNAs are more resistant to degradation and exhibit greater stability under environmental stressors. These characteristics suggest that microRNAs hold significant potential in forensic applications. However, comprehensive studies on miRNA stability in forensic contexts remain limited. The aim of this study was to investigate the time-dependent stability of miR-144, miR-451, and miR-16 miRNAs, as well as SNORD48, in blood stains stored at room temperature (22–25°C) and at −20°C. For this purpose, blood samples were collected from six different volunteers and stored for up to 16 months under defined conditions. Subsequently, RNA isolation, cDNA synthesis, and real-time quantitative PCR (RT-qPCR) were performed on the biological samples. In the study, miR-144, miR-451, and miR-16 miRNAs were used as target markers, while SNORD48 was used as an internal control. Statistical evaluations of the miRNA expression levels obtained at the end of the study were conducted using SPSS version 27. Descriptive analyses and visualizations were performed using the R packages rstatics and ggplot2. When the Ct values obtained from fresh blood samples were compared with those stored at room temperature (22–25°C) and −20°C, it was observed that the expression levels of all three miRNAs significantly decreased after the 8th month. This decrease was more pronounced in samples stored for up to 16 months and was statistically significant compared to fresh samples. Significant statistical differences were observed between the expression levels, particularly for the internal control SNORD48, depending on the storage conditions. These results indicate that SNORD48 a reliable reference gene for miRNA analysis in degraded samples. Although the selected miRNAs remained stable at −20°C, the results indicate that in case of storage at room temperature, miRNA expression is negatively affected due to degradation or exposure to environmental factors. Therefore, careful consideration should be given to the use of blood samples in miRNA analysis and identification processes. Keywords: Forensic, RNA degradation, miRNA, blood identification, miRNA stability