Selection and Validation of Endogenous Reference microRNAs for Post-Mortem Interval Estimation in Vitreous Humor: A...

Area of Science:

• Forensic pathology
• Molecular biology
• Biochemistry

Background:

• Accurate post-mortem interval (PMI) estimation is crucial in forensic pathology.
• MicroRNAs (miRNAs) in vitreous humor (VH) show promise for PMI determination.
• Quantitative Real-Time PCR (qPCR) requires validated endogenous reference genes for reliable results.

Purpose of the Study:

• To identify and validate a stable endogenous reference gene for miRNA analysis in vitreous humor.
• To assess the suitability of miR-222-3p and hsa-miR-96-5p as reference genes.
• To improve the reliability of molecular thanatochronology.

Main Methods:

• Comparative validation of two candidate reference genes (miR-222-3p and miR-96-5p) in 47 forensic autopsy cases.
• Assessment of amplification detectability, expression stability (CV), and statistical independence from PMI and pre-freezing time.
• Application of regression models to analyze gene expression stability.

Main Results:

• MiR-222-3p showed poor detectability, failing the threshold in 61.7% of samples, rendering it unsuitable.
• Hsa-miR-96-5p demonstrated high detectability and expression stability (CV = 9.07%).
• Hsa-miR-96-5p showed no significant correlation with PMI (p=0.69) or pre-freezing time (p=0.70).

Conclusions:

• MiR-222-3p is not a suitable reference gene for miRNA analysis in forensic vitreous humor.
• Hsa-miR-96-5p is validated as a robust and stable endogenous reference gene for vitreous humor.
• Adoption of hsa-miR-96-5p is recommended to standardize molecular thanatochronology and enhance PMI estimation accuracy.