CircRNA circRtn4 as a candidate molecular marker for postmortem interval estimation in mouse brain tissues

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Introduction Estimation of the postmortem interval (PMI) remains a major challenge in forensic research. Traditional PMI estimation methods using postmortem changes were usually inaccurate or empirical. Owing to their stability, abundance, and tissue specificity, circular RNAs (circRNAs) are promising candidates for PMI estimation. This study explores circRNA markers and aims to develop PMI estimation models in mouse brain tissue, with exploratory modeling in human samples. Methods Candidate circRNAs and reference genes were selected from online databases, and primers were designed accordingly. The back-splice junctions of candidate circRNAs were detected by Sanger sequencing. Total RNA was extracted from mouse and human samples collected across different PMIs and temperatures. Semi-quantitative RT-PCR and RT-qPCR were used to quantify circRNAs’ levels, followed by linear and nonlinear regression modeling. Predictive accuracy was assessed using validation samples. Results At 25 °C, mRNA degraded faster than the other tested RNAs. According to postmortem stability, 28S rRNA, mt-co1, and circCDR1as were selected as reference genes for the PMI estimation model at room temperature, and 28S rRNA and mt-co1 were selected as reference genes at high temperature. CircRtn4 exhibited PMI-associated changes under both experimental conditions. In semi-quantitative RT-PCR experiments, model comparison supported quadratic models for the 25 °C data normalized to 28S rRNA and circCDR1as, whereas linear models were favored for mt-co1 at 25 °C and for both internal reference at 35 °C. Additionally, cubic terms provided no additional benefit. Validation using known-PMI samples showed variable prediction errors across models and time points. In the RT-qPCR analysis at 25 °C, only the linear model showed a statistically significant relationship with PMI. Exploratory regression equations were fitted to the single-donor human data at 20 °C and 25 °C. Discussion CircRtn4 may serve as a molecular marker for PMI estimation in mouse brain tissue under the tested temperature conditions. These findings indicate that postmortem circRNA-based models should be developed under defined temperature and species conditions. Further independent validation in veterinary-relevant species and human cohorts is required before considering forensic application.

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Related topics: Molecular Biology Techniques and Applications · Forensic Entomology and Diptera Studies · Circular RNAs in diseases