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

Overview

This study addresses a methodological gap in forensic miRNA-based post-mortem interval estimation by validating endogenous reference genes for qPCR normalization in vitreous humor samples. The research evaluated two candidate normalizers, miR-222-3p and miR-96-5p, across 47 autopsy cases with PMIs spanning 3-24 hours to establish a standardized approach for molecular thanatochronology.

Methods and approach

Vitreous humor specimens were collected from 47 forensic autopsy cases and analyzed using quantitative Real-Time PCR. Two reference gene candidates were subjected to comparative validation assessing detectability (Cq threshold <35), expression stability via coefficient of variation, and independence from biological and pre-analytical variables. Linear regression models evaluated correlations between candidate gene expression levels and both post-mortem interval and pre-freezing storage duration.

Key Findings

MiR-222-3p exhibited poor detectability with failure to achieve sufficient amplification (Cq <35) in 61.7% of samples (29/47), disqualifying it as a suitable normalizer. In contrast, hsa-miR-96-5p demonstrated robust performance with high detectability across valid samples and low expression variability (coefficient of variation 9.07%). Linear regression analysis confirmed statistical independence of hsa-miR-96-5p expression from both PMI (p=0.69) and pre-freezing interval (p=0.70), establishing its suitability as a stable reference gene.

Implications

The validation of hsa-miR-96-5p addresses a critical technical requirement for standardizing qPCR-based PMI estimation methodologies in vitreous humor analysis. This endogenous reference gene enables normalization of amplification data independent of decomposition-related degradation, improving the reproducibility of molecular thanatochronology assays across different laboratories and case conditions. Implementation of this validated normalizer is expected to enhance the reliability and accuracy of forensic PMI estimation models incorporating miRNA biomarkers.