COMET ASSAY INTEREST GROUP

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Comet Assay as a regulatory tool:

Scientific and GLP perspective

 

Single cell get electrophoresis or the Comet Assay has potentials to be used as a second in vivo assay for assessing mutagenic potentials of chemicals.  Before it can be used routinely, the following issues need to be considered.

Sampling issues:  Various tissues are mechanically disrupted to produce single cell suspensions.

Effect of sample preparation on DNA migration.

Contamination with blood cells. eg. Contamination of blood cells when isolating lung cells.

Control on selection of correct anatomical sites sample preparation.  eg. Which layer of stomach?

Should we harmonize the protocol for single cell preparation?

Experimental issues: This is very important when a large number of samples are analyzed in a single experiment.

Lysis step: Does the duration of lysis have any effect on the DNA migration, or cloud formation? Does the number of clouds disappear with increasing lysis time? Previous attempts to address these issues have given conflicting results.

Electrophoresis step: Effect of relative position of slides in the electrophoresis tank? Differential migration of DNA within the slide?

Scoring issues:

Is 50 cells enough for making a reasonable interpretation?  The answer is ‘no’. In our hands, we need a minimum of 400 cells to get fairly consistent data.

How could one incorporate the % clouds with % DNA migration?  What is the relationship between the 2 endpoints? H2O2 sometimes give good DNA migration, whereas at the same concentration it gives rise only to clouds; Why? What is the mechanism?

Manual v/s automated scoring

Data interpretation:

Cytotoxicity and necrosis issues

Pathology issues: How to correlate pathology results with DNA migration?

Can we make the comet assay more specific by using enzymes like FPG and EndoIII?

General issues like how do we reduce variability in comet assay?

 

Please send me your thoughts on these issues.