Evaluation of extraction methods for co-isolation of nucleic acid from human saliva for forensic body fluid identification

MicroRNAs (miRNAs) have emerged as promising biomarkers for the identification of forensically significant body fluids, aiding crime scene reconstruction and the identification of biological material donors. However, despite their potential, inter-study discrepancies and lack of reproducibility have limited their forensic application, particularly in body fluid identification. A major challenge lies in the variability introduced by different nucleic acid extraction methods. This study aimed to evaluate the co-extraction performance and miRNA detection efficiency of three commonly used nucleic acid extraction kits (AccuPrep Genomic DNA Extraction Kit, QIAamp DNA Mini Kit, and miRNeasy Tissue/Cells Advanced Micro Kit) across five saliva input volumes (400 µL, 200 µL, 100 µL, 50 µL, and 25 µL). To assess yield, purity, robustness (CV%), and sensitivity, two quantification platforms were used, followed by a two-step reverse transcription quantitative polymerase chain reaction (RT-qPCR) protocol to evaluate the impact of extraction methods on miRNA expression. Our results demonstrate that both the extraction procedure and the quantity of input material significantly influences nucleic acid recovery. Additionally, we observe variations in miRNA levels depending on the extraction method used. Surprisingly, the kit designed specifically for miRNA extraction yielded relatively poor miRNA recovery. In contrast, the DNA extraction kit (AccuPrep Genomic DNA Extraction Kit) produced the highest nucleic acid yield with moderate purity and showed the lowest Cq values for miRNA targets, indicating better miRNA detection. These findings underscore the importance of the choice of extraction kit, as it can significantly influence both the yield and quality of nucleic acids detected, and the extension, accuracy and reliability of miRNA-based forensic analyses.