Slightly increased level of DNA migration in the comet assay: does statistical significance equal biological...

In the comet assay, DNA damage is assessed by differences in DNA migration from gel-embedded nucleoids. Even a small difference in DNA migration between exposure groups can be statistically significant but may invite speculation about the biological significance of such slight increases in DNA migration. A small difference can be defined as a net difference of 1-2% Tail DNA, but background levels of DNA migration typically vary already more than 1-2% Tail DNA between studies. Here, we have used studies on ionizing radiation to assess the lowest detectable differences in DNA migration; variation in exposure-effect relationships; variation in central tendencies of DNA migration; unsystematic (residual) variation; and the actual number of lesions detectable with the comet assay. A total of 51 studies on ionizing radiation exposure in mammalian cells have been systematically reviewed, including results from ring-trial studies where the same batch of irradiated cells has been analysed in different laboratories. Ring-trial studies have shown that unsystematic variation is approximately 4% Tail DNA in studies on ionizing radiation. Studies on ionizing radiation in cell cultures have shown statistically significant effects when the net increase of DNA migration is 0.3-3.1% Tail DNA. Among those experiments, the ones with optimal assay conditions to detect low levels of DNA damage show statistically significant effects with doses of around 0.30 Gy, which corresponds to approximately 350 lesions per diploid cell. However, it has also been shown that the same dose of ionizing radiation can give rise to different levels of DNA migration (i.e. 0.7-7.8% Tail DNA per Gy) in different studies. In summary, the results show that even a small statistically significant difference in DNA migration has biological significance within the same experiment, but comparisons of DNA migration values between studies have limited biological implications.