Evaluating stability of miR-891a and miR10a markers in semen samples to various conditions

Forensic investigations rely heavily on biological evidence to identify perpetrators and uncover the truth behind criminal activities. Advances in healthcare technologies have made it possible to analyze DNA from biological samples, enabling more definitive and reliable results in legal contexts. Today, before DNA analysis is performed on evidence collected from a crime scene, it is necessary to confirm that the sample originates from a human biological source. Various physical, immunological, histochemical, and microscopic tests have been developed for this purpose. However, when working with trace amounts of samples, these tests can damage the DNA structure and reduce the sample quantity. Furthermore, tests that are not sufficiently specific may yield false-positive or false-negative results. Considering these disadvantages, forensic sciences have turned to next-generation molecular applications. RNA molecules have emerged as alternatives due to their tissue-specific expression characteristics. Like mRNA, miRNAs are also suitable for the identification of bodily fluids due to their tissue-specific expression features. miRNAs are more resilient and stable than mRNAs because of their smaller size and protection by enzyme complexes. Therefore, they produce better results under degraded conditions. Given the degrading environmental conditions at crime scenes, evaluating the stability of such markers in these environments is crucial for forensic sciences. For this reason, the most common crime scene scenarios were determined in our study, and experiments were conducted under these conditions. In our study, the stability of miR-10a and miR-891a, which are markers found in significantly higher concentrations in semen compared to other bodily fluids, was evaluated under degrading conditions. Semen samples were collected from five healthy (normospermic) volunteers. The samples were deposited on 100% cotton white fabrics, which were then exposed to simulated crime scene conditions. As the control group, O-group samples were stored at room temperature. S-group samples were submerged in water obtained from Büyükçekmece Lake, T-group samples were buried in standard garden soil, and Y-group samples were washed in a domestic washing machine at 90 °C with powdered detergent for 1 hour. After the target duration, total RNA was isolated from the fabric samples. Subsequently, cDNA synthesis was performed. Quantitative analysis was carried out using an RT-PCR instrument, where the amplification of miR-10a and miR-891a regions was monitored in real-time, and Ct values were obtained. RNU6b was used as a reference for comparison. For miRNA concentration comparisons, ΔCt [Ct(miRNA) – Ct(RNU6b)] and ΔΔCt values were calculated, and the stabilities of miR-10a and miR-891a were interpreted using the fold-change principle calculated by the 2^-ΔΔCt formula. Samples with Ct values of 35 or higher were excluded from fold-change and statistical analyses. Based on fold-change data, it was observed that miR-891a expression was higher and more stable than miR-10a. In the samples washed in the washing machine, the high temperature and the presence of chemicals such as laundry detergent, which damage the nucleic acid structure, led to greater miRNA degradation. Therefore, miRNA could not be recovered from this group, and comparative analyses were not performed. Statistical analyses were conducted using IBM SPSS Statistics 22 to determine whether different environmental conditions significantly affected miRNA concentrations. Normality was assessed with the Shapiro–Wilk test, while One-way ANOVA and Post-hoc Tukey tests were used for group comparisons. According to the results of the One-way ANOVA test, the tested environmental conditions did not have a significant impact on miRNA stability (p>0.05).