Abstract
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used for nucleic acid quantification. The use of technical triplicates in RT-qPCR aims to minimize variability and improve reliability but increases reagent consumption, labor, and time. This study systematically evaluates the necessity of technical replicates by analyzing 71,142 cycle threshold (Ct) values from 1,113 RT-qPCR runs across three instruments, two detection chemistries, and 30 operators. Variability within replicates was assessed using metrics such as the coefficient of variation (CV), while the impacts of operator expertise, detection chemistry, instrument calibration, and initial template concentration were explored. The findings challenge the assumption that variability increases at low template concentrations, revealing no correlation between Ct values and CV. While inexperienced operators exhibited slightly higher variability, their replicates were still consistent, with acceptable CVs and low outlier frequencies. Dye-based detection showed greater variability than probe-based. Time since calibration had negligible effects on replicate consistency. Notably, duplicate or single replicates sufficiently approximated triplicate means. These results challenge traditional assumptions about RT-qPCR variability and provide a data-driven framework for optimizing experimental design. This study offers potential for resource savings without compromising data quality, particularly in high-throughput applications or laboratories with limited funds. The data underlying this article are available at https://doi.org/10.5281/zenodo.15072870.