Why comet assays tails are not appearing?

15 February 2023 9 2K Report

We did comet assay based on joves protocol. Under fluorescence microscope, cells were visible but without tail. The drugs showed toxicity when grown in 6-well plate. Lysis was done for overnight at 4 degree celsius. Electrophoresis was run at 25V, 300mA for 30mins.

Pankaj Sharma

If you are able to see stained cells under the microscope, and there is no tail formation then probably no DNA damage has been induced by the drug. Toxicity itself does not mean DNA has been damaged. There can be other factors in play.

Ameena Stellixir

Thank you sir. The cell line we used was MCF7. Even the standard group slides (camptothecin) didn't show any tails. Do you have any other protocol that you have done with?

Pankaj Sharma

https://rdcu.be/c5LcN

This is the protocol I follow, try repeating it might work, it's a sensitive protocol.

Ameena Stellixir

Thank you sir. May I know whether you done electrophoresis at 4degree celsius?

Pankaj Sharma

Nope I did not do electrophroesis at 4 degrees, it was done at room temperature only. I was mixing my cells in low melting agar, then coating the slides with the suspension.

Ameena Stellixir

@Pankaj Sharma. Sir can you please specify details regarding the agarose and low melting agarose you have used for comet assay?

Pankaj Sharma

SeaPlaque™ Agarose (Catalog #: 50101, Lonza) and SeaKem® GTG™ Agarose Catalog #: 50070

Ameena Stellixir

Thank you very much sir.

Nikolay Klyashtornyy

If cells are visible but without tail in the comet assay, it usually means that DNA is not fragmented or the level of fragmentation is very low. There are several reasons why comet assay tails may not appear:

Incomplete lysis: Proper cell lysis is essential for the release of DNA from cells. If lysis is incomplete, the DNA may not be fully released, leading to low or no fragmentation.

Overloaded slides: If too many cells are loaded on the slide, the DNA fragments may not be able to migrate far enough from the head to form a tail.

Electrophoresis conditions: The conditions used for electrophoresis, such as voltage, current, and time, can also affect the formation of comet tails. If the voltage is too high, the DNA fragments may migrate too quickly and not have enough time to form a tail.

DNA damage level: If there is little or no DNA damage present in the cells, the comet tails may not be visible.

To troubleshoot the issue, you may want to adjust the electrophoresis conditions, such as lowering the voltage or extending the electrophoresis time, to encourage the formation of tails. It may also be helpful to optimize the lysis conditions to ensure complete DNA release. Additionally, you can try reducing the number of cells loaded on the slide to ensure proper migration of DNA fragments. Finally, it may be helpful to confirm the presence of DNA damage by performing other assays, such as the alkaline or neutral comet assay.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

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