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Challenges in mRNA-based time since deposition estimation: A multiplex MPS primer panel investigation.

Nadescha Viviane Hänggi1, Melisa Walliser1, Ane Elida Fonneløp2

  • 1Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland.

Forensic Science International. Genetics
|January 7, 2026
PubMed
Summary

Estimating time since deposition (TsD) of biological stains using mRNA degradation patterns is promising for forensic investigations. However, variability in markers and methods presents challenges for accurate TsD estimation.

Keywords:
Body fluidsForensic scienceMassively parallel sequencing (MPS)Time since deposition (TsD)mRNA

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Area of Science:

  • Forensic science
  • Molecular biology
  • Genetics

Background:

  • Estimating the time since deposition (TsD) of biological stains is critical for crime scene investigations.
  • Current methods for TsD estimation are limited, necessitating the development of new approaches.

Purpose of the Study:

  • To investigate mRNA degradation patterns in four body fluids (blood, semen, menstrual blood, vaginal secretion) for TsD estimation.
  • To assess the utility of a custom mRNA MPS primer panel for analyzing degradation patterns.

Main Methods:

  • Utilized a custom mRNA MPS primer panel designed with candidate markers identified via RNA-Seq.
  • Analyzed mRNA degradation based on individual trends, intra-transcript stability (5' vs. 3' ends), and inter-transcript stability.

Main Results:

  • Observed significant inter-individual variability in mRNA degradation, particularly for low-abundance transcripts.
  • Identified some mRNA markers whose degradation patterns correlated with TsD.
  • Encountered inconsistencies in degradation patterns, indicating challenges in primer panel optimization, RNA extraction, and reproducibility.

Conclusions:

  • mRNA degradation shows potential for forensic TsD estimation.
  • Forensic application requires careful selection and validation of markers across diverse datasets to address biological and methodological variability.