% DNA in Tail Calculation
This method is based on determining the relative circularity of the comet head and the total comet which may contain a comet tail if there is significant DNA damage. The following equation can be used:
This analysis works well due to the fact that the total comet becomes less circular with increasing DNA damage. In the case of little DNA damage, it is apparent that Total Comet Circularity ≈ Comet Head Circularity, thus % DNA in Tail ≈ 0%.However in the case of extensive DNA damage, Comet Head Circularity >> Total Comet Circularity and % DNA in Tail will tend towards 100%.
We used Gen5 Data Analysis Software to apply this calculation to all images, and using the 96-well CometChip calculations (Table 1) as an example, we can see that % DNA in Tail values increased appropriately with increasing etoposide treatment (T1-T3) of the healthy Alkaline CometAssay Control Cells (T0). The % DNA in Tail values are comparable to those obtained via different methods like ImageJ open source software with the OpenComet plugin.
|Table 1. % DNA in tail calculations for 96-well CometChip|
Gen5 results compare well to those seen using Loats Analysis software using the 96-well CometSlide (Figure 1), further confirming that Cytation 5 and Gen5 deliver accurate results regardless of the configuration.
|Figure 1. Percent DNA in tail calculations for 96 well CometSlide using Alkaline CometAssay Control Cells in the standard comet assay|
Comet Tail MomentAnother comet evaluation calculation is the Comet Tail Moment (Figure 2), which takes into account the Total Comet Length (DS1), Comet Head (DS2) and % DNA in Tail (DS3) as represented in the following equation:
|Figure 2. Comet areas included in comet tail moment calculation|
|Table 2. Comet Tail Moment calculations for 3 well CometSlides.|
As Cytation 5 automates cellular analyses, DNA damage using the comet assay is rapid, repeatable and comparable to analyses obtained via different analyses methods. For more detail, see our application note, Automated Imaging and Analysis of a Novel Comet Assay to Enable High Throughput Genotoxicity Testing.