Abstract
Estimating the post-mortem interval (PMI) using microRNAs (miRNAs) in vitreous humor (VH) is a promising technique in forensic pathology. However, the reliability of quantitative Real-Time PCR (qPCR) data in this matrix is currently constrained by a critical methodological challenge: the lack of a rigorously validated endogenous reference gene (normalizer) capable of correcting for non-biological variations without being influenced by decomposition. This study aimed to identify a robust reference gene for VH analysis by performing a comparative validation of two candidates proposed in the literature: miR-222-3p and miR-96-5p. VH samples were collected from 47 forensic autopsy cases with estimated PMIs ranging from 3 to 24 h. The validation process assessed three key parameters: amplification detectability, expression stability (Coefficient of Variation, CV), and statistical independence from both the PMI and the pre-analytical freezing interval using regression models. MiR-222-3p was rejected as a normalizer due to poor detectability, failing to reach the detection threshold (Cq < 35) in 61.7% of cases (29/47). Conversely, hsa-miR-96-5p was validated as a stable reference gene. It demonstrated high detectability and expression stability (CV = 9.07%) among valid samples. Crucially, linear regression analysis showed no significant correlation between hsa-miR-96-5p levels and either the PMI (p = 0.69) and the pre-freezing time (p = 0.70). This study demonstrates that miR-222-3p is unsuitable for forensic casework in VH due to instability. We identified and validated hsa-miR-96-5p as a robust endogenous reference gene. Its adoption is recommended to standardize future molecular thanatochronology studies and improve the accuracy of PMI estimation models.