Assessing the necessity of technical replicates in reverse transcription quantitative PCR.

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 templat

SUBMITTER: Christoforidou E 

PROVIDER: S-EPMC12315840 | biostudies-literature | 2025 Apr

REPOSITORIES: biostudies-literature