PCR-Stop analysis as a new tool for qPCR assay validation.

ABSTRACT: Progressively more qPCR assays have been developed in recent years in numerous fields of application. These assays are routinely validated using calibration curves, but essential validation per se such as Poisson analysis is frequently neglected. However, validation is crucial for determination of resolution and quantitative and qualitative limits. The new test method PCR-Stop analysis presented in this work investigates assay performance during initial qPCR cycles. PCRs with one to five pre-runs are performed while the subsequent main qPCR runs reflect pre-run replication rates. Ideally, DNA doubles according to pre-runs, there is no variation between replicates and qPCR starts immediately at the first cycle with its average efficiency. This study shows two exemplary qPCR assays, both wit

SUBMITTER: Witte AK 

PROVIDER: S-EPMC5974342 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature