Forensic evaluation of the reliability and validity of XIST and RPS4Y1 RNA biomarkers as sex discriminators for touch...

The forensic determination of biological sex from trace evidence remains challenging because of nucleic acid degradation in touch samples. Recent advances suggest that RNA, particularly sex-specific markers such as X inactive specific transcript (XIST) and ribosomal protein S4 Y-linked 1 (RPS4Y1), may serve as more reliable alternatives.

This study aimed to assess the reliability and temporal stability of these markers across different surfaces and time intervals. Touch samples from male and female donors were deposited on glass and leather, then analyzed immediately, after 14 days, and after 28 days. Quantitative real-time PCR was used to measure XIST (female-specific) and RPS4Y1 (male-specific), normalized to GAPDH. The results demonstrated consistent sex-specific expression, with XIST robustly expressed in female samples and RPS4Y1 strongly expressed in male samples, while absent in females. Both markers were detectable up to 28 days, although surface-dependent differences were noted. Leather preserved RNA better than glass, with RPS4Y1 showing greater degradation on glass, whereas XIST remained stable on leather. Significant temporal and surface effects were observed (p < 0.0001), yet both genes retained discriminatory power over time.

These findings validate XIST and RPS4Y1 as reliable RNA biomarkers for sex determination in forensic touch samples. Their specificity and durability, particularly on leather surfaces, highlight their potential integration into routine forensic workflows, providing investigators with a robust method for determining the biological sex of individuals from minimal and degraded biological material.