The 5' GAPDH transcript region as a stable endogenous reference for postmortem RT-qPCR in human thyroid gland and skeletal muscle: proof of concept.

Accurate estimation of the postmortem interval (PMI) remains one of the major challenges in forensic medicine. Recent advances in molecular biology have introduced novel approaches for the evaluation of mRNA degradation; however, reliable interpretation of postmortem data requires a stable endogenous reference for normalization. Using real-time quantitative PCR (RT-qPCR), we analyzed GAPDH mRNA degradation in psoas major muscle and thyroid gland tissues through two experimental approaches: (1) time- and temperature-stability experiments and (2) analysis of cadaveric samples collected at different PMIs. For this purpose, we used our in-house-developed probe targeting the 5' region of the GAPDH mRNA. The obtained data demonstrated relative stability of GAPDH mRNA in both tissue types regardless of storage temperature. Overall, our findings indicate high stability and reproducibility of the applied assay, supporting the potential use of the targeted 5' GAPDH region as an endogenous reference for normalization in postmortem RT-qPCR studies in forensic and legal medicine.