With increased use of real-time polymerase chain reaction technology in molecular
diagnostics, consistent procedures for design, optimization and validation of molecular diagnostic
methods are needed. This chapter describes a practical guiding principle that can be used in different
steps of the design and validation of in-house developed real-time PCR assays. The use of the described
guidelines leads to more efficient and standardized optimization and validation. Ultimately, this results
in a reliable and robust molecular diagnostic assay. A statistical follow-up of the performance of the
assay is included and can be achieved by determination of target values and reproducibility of internal
quality controls. Since this guiding principle is independent of environment, equipment and specific
applications, it can be used in any laboratory.
Keywords: Oligonucleotide design; in-house assay; target gene; optimization; validation; specificity;
melting curve analysis; amplicon sequencing; linearity; annealing temperature; internal quality control.
