Internal and External Controls for Reagent Validation

PCR applications that require a high confi dence in the result should be designed to control for the occurrence of false negatives. False negatives can occur from inhibition of one or more of the reaction components by a range of factors. While an external, or batch control is often used, the ideal control is one that is included in the reaction cocktail in a multiplex format. Early approaches used different sized amplicons combined with end-point analysis. Fluorescent homogenous real-time PCR methods have a number of advantages for implementing internal controls. Here we discuss the application and development of molecular mimics for use as controls in real-time PCR, and explain a number of concepts and experimental considerations that will aid in the optimisation of the controlled multiplexed assay. caister.com/pcrbooks