Tuesday, January 12, 2016

Beyond the Solar System: Automated Comet Analysis

In our last comet assay blog post, ‘Beyond the Solar System: Automated Comet Assay Imaging’, we discussed how the comet assay  is used to directly quantify DNA damage in mammalian cells, and showed how to use Trevigen’s CometAssay® Electrophoresis System, 3-Well FLARE™ slides and 96-Well CometSlides with the Cytation 5™ Cell Imaging Multi-Mode Reader to easily image these comets. Here we’ll discuss how to analyze those images to quantify the extent of DNA damage. This quantification can be expressed as either the % DNA in the Tail or the Comet Tail Moment. Each method quantifies DNA damage by assessing the fluorescence from comet tails attributable to DNA fragments electrophoresing farther in the gel than intact DNA.

% 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:
DNA in tail
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.
% DNA in tail calculations for 96-well CometChip
Table 1. % DNA in tail calculations for 96-well CometChip

comet head primary cellular analysis
 
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.
Percent DNA in tail calculations for 96 well CometSlide using Alkaline CometAssay Control Cells in the standard comet assay
Figure 1. Percent DNA in tail calculations for 96 well CometSlide using Alkaline CometAssay Control Cells in the standard comet assay

Comet Tail Moment
Another 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:

(DS3*(((DS1-DS2)/2)+(DS2/2)))/100
 
Comet areas included in comet tail moment calculation
Figure 2. Comet areas included in comet tail moment calculation
 
Comets exhibiting little to no DNA damage will have a Comet Tail Moment value approaching zero, whereas those with higher damage amounts will have increasing values. Gen5 Data Analysis Software was again used for automatic calculations, and as seen in Table 2, the Comet Tail Moment values increased appropriately with increasing etoposide treatment (CC1-CC3) as compared to untreated cells. Furthermore, these results are equivalent to those obtained via accepted methods like Loats Analysis software.

Comet Tail Moment calculations for 3 well CometSlides.
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.
 
 
By, BioTek Instruments

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