Evaluation of Reduced-Volume qPCR Reactions for Forensic DNA Quantification Using the Investigator Quantiplex Pro RGQ Kit

Background: The Investigator Quantiplex Pro RGQ Kit (Qiagen, Hilden, Germany) enables rapid quantification of total human and male DNA on the Rotor-Gene Q platform while simultaneously assessing PCR inhibition, DNA degradation, and mixed DNA profiles. This study evaluated whether reducing the recommended qPCR reaction volume by half affects analytical performance, with the aim of improving throughput and reducing reagent costs in forensic laboratories. Methods: Control DNA (50 ng/µL) and DNA extracted from 20 simulated forensic samples representing common forensic evidence (including bloodstains, semen stains, saliva samples, vaginal swabs, touch DNA, hair roots, and mixed biological traces deposited on different substrates) were analyzed using standard and half-volume qPCR reactions with identical reagent proportions. Quantification and quality indices—including inhibition (IC), degradation (DI), and mixture (MI)—were evaluated using Q-Rex software. Ct values, ΔCt, and standard deviations were compared between protocols using paired statistical analysis, with significance set at p < 0.05. Results: Standard curves generated under both reaction conditions showed efficient amplification, with R2 values > 0.998 and slopes within recommended ranges. Control samples demonstrated strong agreement between expected and measured DNA concentrations for both protocols. Although forensic samples displayed variable DNA quantity and quality, highly concordant results were obtained between standard and reduced volumes. Mean Ct values showed minimal ΔCt variation and low dispersion across all sample types. IC, DI, and MI indices were also consistent, indicating reliable detection of inhibition, degradation, and mixed DNA profiles under reduced-volume conditions. Conclusions: Half-volume qPCR reactions using the Investigator Quantiplex Pro RGQ Kit maintained accurate, precise, and reproducible DNA quantification and quality assessment. Reduced-volume protocols may therefore provide a cost-effective strategy to increase throughput and improve workflow efficiency in forensic laboratories without compromising analytical robustness.