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Temporal RT-qPCR-Based Porcine Cardiac Molecular Profiling for Post-Mortem Interval Estimation: Predictive Modeling.

Vincenzo Cianci1, Cristina Mondello1, Francisco J Diaz2

  • 1Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Section of Legal Medicine, University of Messina, Via Consolare Valeria, 1, 98125 Messina, Italy.

International Journal of Molecular Sciences
|June 12, 2026
PubMed
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This summary is machine-generated.

Estimating post-mortem interval (PMI) using RNA markers is challenging. While cardiac gene expression shows temporal trends, reference gene stability and analytical methods limit predictive accuracy for PMI estimation.

Area of Science:

  • Forensic Pathology
  • Molecular Biology
  • Bioinformatics

Background:

  • Estimating post-mortem interval (PMI) is crucial in forensic investigations, especially after the early postmortem period.
  • RNA-based markers analyzed via quantitative real-time PCR (RT-qPCR) offer potential for PMI estimation.
  • Reliability hinges on technical consistency, stable reference genes, and appropriate data analysis.

Purpose of the Study:

  • To evaluate the utility of postmortem cardiac gene expression for PMI estimation.
  • To assess the stability of reference genes and the performance of different analytical models.
  • To identify informative RNA markers for temporal profiling.

Main Methods:

  • Nine porcine hearts were refrigerated (4°C) and sampled over 120 hours.
Keywords:
PMI estimationforensic molecular pathologymRNA degradationpostmortem predictive modelingreference gene stability

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  • RT-qPCR was performed on target genes (BAX, CASP3, HIF1A, HMOX1) and reference genes (ACTB, RPL4, GAPDH, HPRT1).
  • Statistical analyses included mixed-effects models and leave-one-out cross-validation, comparing raw Ct, ΔCt, and ΔΔCt values.

    • Main Results:

    • Technical reproducibility was satisfactory, but reference genes showed temporal instability (RPL4 drifted).
    • HPRT1 and HMOX1 were the most informative markers for PMI.
    • Models using raw Ct values outperformed ΔCt models; a parsimonious model with HMOX1 and HPRT1 showed promise but had non-negligible error.

    Conclusions:

    • Postmortem cardiac gene expression contains temporal information for PMI modeling.
    • Predictive performance is constrained by reference gene behavior, analytical strategy, and inherent estimation errors.
    • Further validation in real-world forensic settings and methodological standardization are essential for clinical application.