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.
Lazzari et al. (Tue,) studied this question.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: