Stability in question? A preliminary ddPCR-based study on the stability of 14 miRNAs in five forensic body fluids

Abstract

MicroRNAs (miRNAs) have emerged as one of the most valuable biomarkers in forensic applications owing to their biological information (e.g., tissue-specific expression) and high stability—an enabling property that preserves their utility in challenging forensic samples. While previous studies have demonstrated that the quality and degradation of mRNA and lncRNA can significantly affect gene expression profiling, the degradation dynamics of miRNAs in forensic body fluids remain underexplored. Droplet digital PCR (ddPCR), with its high sensitivity and precision, enables the detection of low-abundance targets and subtle changes in gene expression, thus providing a robust platform for analyzing RNA degradation patterns. In this preliminary study, we utilized RNase A digestion across time gradients to simulate varying degrees of RNA degradation in five forensically relevant body fluids. Using ddPCR, we investigated the degradation patterns and influencing factors of 14 miRNAs previously proposed for body fluid identification. Most miRNAs exhibited biphasic decay kinetics; however, a subset showed unexpected increases in concentration over time. Regression analysis revealed that miRNA degradation rates are influenced by multiple factors: specifically, miRNAs with higher predicted free energy tended to degrade faster; higher initial expression levels were associated with slightly slower degradation; and the body fluid-specific microenvironment may exert a protective effect through protein-mediated stabilization. These findings highlight the heterogeneity of miRNA degradation, which may introduce analytical bias in degraded forensic samples. Future studies should expand the evaluation of miRNA degradation characteristics to identify degradation-resistant biomarkers and develop correction strategies to minimize the impact of RNA degradation on gene expression analysis.