In the current study, laboratory efforts to prevent the presence of positive NTCs (no template controls) during the optimization of a quantitative real-time reverse transcriptase PCR assay for detection of Noroviruses (NoVs) are described. Two DNA types (single-stranded (ss)DNA fragments and plasmid DNA) were used to generate a real-time PCR standard and a high frequency of positive NTCs was noticed in the case of ssDNA fragments. To investigate our suspicion of well-to-well migration of DNA during real-time PCR runs as possible cause of the positive NTCs, an "evaporation-experiment" was set up in which the evaporation of water and the possible co-evaporation of DNA were measured as a function of the DNA type (ssDNA-fragments, plasmid DNA and genomic DNA), the reaction plate seal type (adhesive film or 8-cap strips) and the use of 7 microl of mineral oil as cover layer. Results of this experiment indicated that evaporation of water occurred during real-time PCR runs regardless of the DNA type, the seal type and whether or not 7 microl of mineral oil was used as cover layer. Data from this experiment also suggested co-evaporation of DNA, with an apparent negative correlation between the size of the DNA type and the extent of this co-evaporation. The use of 7 microl of mineral oil as cover layer seemed to prevent to some extent co-evaporation of DNA. The use of plasmids as standard combined with 7 microl of mineral oil as cover layer in the real-time PCR setup resulted in a complete absence of positive NTCs while only minor effects were noticed on the performance of the real-time PCR. In general, our results showed that the high sensitivity of an optimized real-time PCR assay should be considered as--besides a great advantage--a potential risk factor for obtaining false-positive results when using this technique.