The presence of tails in control samples could depend on the type of cells you are using. Are you using a cancer cell line that experiences replication stress for example? Or there could be another type of endogenous DNA damage that causes the tails. Which cell type are you using?

One of my colleauges had tails in his commet results. He named it unclassical effect. According to him, the classical results need to be looking like a fuzzy circle around the cell. So I think it is acceptable to have tails depending on the treatment you are using. You should be able to calculate the fluorescence intensity of your tails as will as the classical form.

The presence of tails in control samples could depend on the type of cells you are using. Are you using a cancer cell line that experiences replication stress for example? Or there could be another type of endogenous DNA damage that causes the tails. Which cell type are you using?

Hi Sean,

If you like, I can have a look at your protocol. I have been doing comet assays for years now and have encountered and dealt with most issues with the assay. Although I never have seen reverse tails. This would indicate that there might be an issue with lysis.. Have a look at the protocol in the M&N of the following paper: Wijnhoven et al., Mol Cell (2015). There the procedure is described in detail. Good luck

The increased tail moment may be caused by DNA damage and other reasons increasing migration of intact DNA loops. Direct prove that increased comets as measured with neutral comet assay may not be dealt with DNA damage was provided by different research groups (Belyaev, Eriksson et al. 1999; Afanasieva, Zazhytska et al. 2010). In these studies, it was shown that increased or decreased neutral comets were caused by decondensation or condensation, respectively, of intact DNA loops. The increased tails measured with alkaline comet assay may be caused by increased number of open replicons or transcription units due to inhibition of their prolongation but not by DNA damage. Due to dependence of control tails on cell type (degree of supercoiling and length of DNA loops, level of transcription and replication), longer or shorter tails can be observed in different cells types

The opposite movement of tail means that the material is positively charged and, thus, it represents a complex of DNA with positive substances such as basic polypeptide/proteins and ions. It should reflect methodological problems with lysis or electrophoresis.

Afanasieva, K., M. Zazhytska, et al. (2010). "Kinetics of comet formation in single-cell gel electrophoresis: Loops and fragments." Electrophoresis 31(3): 512-519.

Belyaev, I. Y., S. Eriksson, et al. (1999). "Effects of ethidium bromide on DNA loop organisation in human lymphocytes measured by anomalous viscosity time dependence and single cell gel electrophoresis." Biochimica et Biophysica Acta (BBA) - General Subjects 1428(2-3): 348-356.

Hi, are you using kit or traditional method?

Based on my experience with traditional method, the tail in untreated group should comes from two major reason: 1. cell type specific and 2. time for experimental procedure.

Some cell lines have tails due to replication stress, but this is rare for an untreated cell to have tails in both neutral and alkaline comet assay. You should do another cell lines as a control to check whether the tails are cell line dependent.

Most of the time, a successful comet assay comes from people who are familiar with the whole experimental procedure. You have to control the time of trypsin treatment, and the time it takes from trypsin to finishing 3 layers and immersing into lysis buffer. In my previous training, we control the trypsin process to within 10 min and finish 3 layers within 15 min to minimize DNA damage during experimental process.

You can try to pull up a published paper which shows no tail in control group and use the same cell line as your "procedure control" if you are a beginner of comet assay. 

(P.S. I keep doing comet assay for more than 5 years and I usually train junior students by repeating published data. Normally, it takes around 3-6 month for them to be independent and reliable comet assay performers.)

A "wrong direction" tail may comes from some positively charged macromolecules on DNA. Check your lysis buffer and electrophoresis buffer, you might want to make a new bottle.

Good luck!

Thank you all for your help! I am new to the comet assay so hopefully it is new user errors that are causing problems. We are also using culture samples taken from animals in the field and since there is no baseline for any amount of DNA or cellular damage we might be dealing with higher than expected numbers. Thank you again!

Dear Sean Raph,

I think that tail comes from some macromolecules on the DNA positively charged. Check your lysis buffer and the electrophoresis buffer, you may want to make a new bottle. Best regards

Our controls were from samples that had a high background >30% DNA damage, we confirmed by staining metaphases and scoring the damage.

Even I find comet tails going in all directions.I would like an answer please.my basic procedures are those of Singh et al and Dhawan et al for flourescence comet assay

Paul WG Wijnhoven seeing your comment for using the comet assay for years I hav query too plz. Could you please help me how to interpret the excel results in my thesis....I have used open comet software...single gel per sample...I am confused for whether to take the mean given in the excel output or the median..sometimes for single image,per sample a single comet is detected with zero SD..nd mean....or i need to apply some sort of statistical test...Kindly guide me