Time Since Death Estimation via <scp>mRNA</scp> Expression of <scp>GAPDH</scp> and <scp>ACTB</scp> in Human Skeletal Muscle

Meticulous determination of the time since death (TSD) is critical in certain criminal investigations. This study aimed to identify optimal markers for TSD estimation and develop a mathematical model applicable under varying conditions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-Actin (ACTB) mRNA levels were analyzed in 10 human skeletal muscle samples using real-time quantitative PCR at eight time intervals (6, 12, 18, 24, 30, 36, 42, and 48 h) within 48 h postmortem at 25°C. Among the candidate markers, GAPDH demonstrated the strongest correlation with times since death. At the same time, ACTB expression remained comparatively stable across all samples and time points as identified by RefFinder (reference gene analysis tool). Mathematical equations were developed using the C_t values for GAPDH, ACTB, and ΔC_t (GAPDH-ACTB) and the cubic model gave the highest determination of coefficient while the error rates were low in the quadratic model. The findings indicate that ACTB is an optimal marker for estimating TSD within the 30 to 36-h postmortem interval, while GAPDH is more suitable for the 36 to 48-h period.