Comet assay - segmentation problem

cometassay

#1

hi all,

I’m new to comet assay and I’m trying to use the comet assay pipeline in cell profiler to automate analysis.
The pipeline is able to identify the primary objects (comets).
However it is unable to demarcate the comet head.
Please suggest how to tweak the pipeline so that I can automate comet head identification.
Alternatively is the problem in my images and not the pipeline?


Any help would be appreciated.
I have uploaded some sample images.

Thanks.


#2

Hi,

In your case, the comet shapes are not that typical, so our typical example “Comet assay” pipeline wouldn’t work for you.

As your comet’s heads and tails have near identical intensities, I suggest to do a segmentation for a whole comet first (head + tail), then use a declumping method “Shape - Shape” to split a head and a tail. You can tweak the “Distance between local maxima” and “Size of smoothing filter” for better separation between heads & tails.

If possible, it is better to improve your laboratory experiment instead. For example, I suggest to set your electrophoresis tank to lower Voltage (not higher 100 V), and allow the assay to run longer.
If you use an alkaline buffer, you can let your sample sit inside this buffer for about 10 minutes without electricity, to allow DNA to unwind before actually pulling them with electric current.

Good luck.


#3

Hi Minh,

Happy New Year!
Thank you so much for your input. I’ll try your suggestions. I did use alkaline buffer and let the gel sit in it for >1 hr before electrophoresis. I ran the gel at 19V which is ~ 1V/cm in my system (distance measured between the electrodes) for a total of 30 minutes.