Quantitative DNA/RNA fragmentation assays for estimating the time since deposition (TsD) of bloodstains

The time since deposition (TsD) of bloodstains can provide crucial information for forensic casework, yet reliable estimation methods remain limited. This study aimed to identify an optimal quantitative PCR (qPCR) approach for bloodstain TsD estimation, analyze factors influencing nucleic acid stability, and construct human-applicable inference models. A total of 244 human bloodstain samples (238 for marker evaluation and model construction, and 6 [T1-T6] for independent validation) were prepared under different time points, temperatures, and substrates (grouped into two sample sets), together with 11 animal samples for primer species-specificity testing. Six candidate markers were initially selected from the literature and further screened based on LinRegPCR analysis, species-specificity tests, and standard-curve evaluation; their stability was then assessed using multiple reference-gene stability tools. Correlation analysis, linear regression, and curve fitting were used to construct and validate the models. Four qualified markers were retained, among which COX1 and 16S rRNA were confirmed to be human-specific. Under the conditions examined, short amplicons of the target genes exhibited expression stability comparable to that of conventional internal reference genes, providing, to our knowledge, the first experimental evidence for an assumption previously adopted without formal verification. Among the four qPCR-based quantification indicators compared, the same-marker relative expression ratio (RER) method showed the strongest correlation with TsD and reduced sex-related inter-individual variation. Nucleic acid stability was jointly affected by marker type, fragment length, storage time, temperature, substrate, and sex, with storage time being the dominant external factor. Eight models demonstrating superior performance were selected (two for Sample Set 1 at 25 °C, and six for Sample Set 2, with two per substrate across blood card, cotton swab, and gauze), all yielding R² > 0.6 for short-term (≤ 2 months) and R² > 0.8 for long-term (up to 2 years) samples, with all mean absolute percentage errors (MAPEs) below 30% on T1-T6. The same-marker RER method is thus proposed as an effective qPCR-based approach for forensic bloodstain TsD estimation.