Molecular forensics: RNA degradation as a marker for postmortem interval determination.

Determining the time since death (TSD) is crucial in forensic investigations, yet it remains a complex process influenced by numerous postmortem factors. This study explores RNA degradation as a promising marker for precise TSD estimation, leveraging real-time quantitative polymerase chain reaction to quantify degradation rates. We analysed degradation patterns of messenger RNA (mRNA), ribosomal RNA (rRNA), and microRNA (miRNA) across liver, lung, and heart tissues, focusing on housekeeping genes (GAPDH, ACTB, RPS10, RPS29) and rRNA (5 S, 18 S, 28 S), alongside tissue-specific miRNAs (miR-122, miR-195, miR-200c, miR-1, U6). RNA yield, and purity varied significantly among tissues, with the liver showing higher RNA yields but lower purity compared to the heart and lung. RPS29 and RPS18 emerged as stable reference markers, unlike GAPDH and ACTB, which showed postmortem instability. rRNA demonstrated greater resilience to degradation compared to mRNA, with distinct patterns peaking at 60-84 h postmortem. miRNAs, particularly miR-122, and miR-1, showed potential as TSD biomarkers due to their relative stability. A mathematical model for TSD estimation was developed using 14,400 Ct values from 600 tissues analyzed in R-studio. The model demonstrated high to moderate predictive accuracy across three tissues under controlled environmental conditions. The model achieved the highest accuracy in heart (R² = 0.75), followed by liver (R² = 0.739), with the lowest predictive power observed in lung (R² = 0.57). These findings underscore the effectiveness of RNA degradation profiles, particularly rRNA and microRNAs, in TSD estimation and highlight the importance of integrating multiple tissues and tissue-specific markers for enhanced forensic reliability.