Real-time PCR: duplexing without optimization.

Abstract

Real-time PCR applications generally require determination of the threshold cycle of a gene of interest in parallel with an endogenous control. In standard situations, the gene-specific and control reactions are run as separate samples (singleplex). In contrast, duplex approaches combine both reactions within a single well, thereby saving time, cost, and material. However, establishing duplex reactions usually requires laborious optimization justifiable only for the analysis of large sample series. Hence, in research settings, singleplex approaches are used most commonly. To establish conditions for duplexing without the need of optimization, we tested the performance of 40 premade TaqMan gene expression assays in duplex reactions with an endogenous control using three different polymerase mixes. The results were compared with singleplex reactions. Duplexing results obtained with one of the multiplex polymerase mixes correlated extremely well (r(2)=0.95) with the singleplex reference. The findings of our study demonstrate that the combination of this polymerase mix and premade gene expression assays will yield reliable and reproducible results in duplex approaches without preceding optimization.