Establishment of molecular diagnostics offering quantitative technology is directly associated with real-time polymerase chain reaction (PCR). This rapid, accurate and sensitive method requires careful execution, including reliable calibration standards. The storage of such standards is crucial to prevent nucleic acid decay and to ensure stable results using real-time PCR. In this study, a broad investigation of possible causes of DNA degradation during storage was performed, including GC-content of the fragments, long-term storage, rapid freeze-and-thaw experiments, genomic DNA and short DNA fragments of different species, the influence of shear stress and the effect of nuclease remaining after DNA isolation. Several known chemical DNA degradation mechanisms have been matched with the experimental data through a process of elimination. Protocols for practical application, as well as a theoretical model describing the underlying mechanisms of deviation of real-time PCR results due to decay of standard DNA, have been developed. Primary amines in the buffer composition, which enhance depurination of the DNA helix, and shear stress due to ice crystal formation, could be identified as major sources of interaction. This results in degradation of the standard DNA, as well as in the probability of occurrence of mismatches affecting real-time PCR performance.