Tail profile: a more accurate system for analyzing DNA damage using the Comet assay.
The Comet assay (single cell gel electrophoresis assay) measures DNA strand breaks in individual cells. In the assay cells are embedded in agarose, lysed, and electrophoresed under low voltage, allowing migration of damaged DNA. The DNA is stained and subsequently viewed with an epifluorescent microscope. If DNA damage has occurred the electrophoresed DNA fragments appear as a diffuse tail behind the nucleus known as a "comet". Many computer-aided analysis systems are currently in use to quantify the amount of DNA damage that is represented by a comet image. Here, we present a novel method of analysis known as "tail profile". This method of analysis provides several advantages over currently employed methods, which rely primarily on the "tail moment" method of analysis. We compared the amount of DNA damage reported from both the tail profile and tail moment methods of analysis and observed a 26% (P<0.0001) increase in damage detected by tail profile across the 10-25 microm range of tail length, where the majority of the relevant comet data is concentrated. We further report that this increase in sensitivity is not only limited to assessing DNA damage, but also to gathering data from DNA repair assays. Furthermore, we demonstrate increased functionality and extended data analysis capabilities with the use of a compressed collection of images called a "comet chip" and through a visual representation of data called a "profile plot". Use of the custom macros enabled us to detect an unexpected characteristic of the electrophoretic profile, giving us novel insight into the nature of comet analysis. In addition to the increased analytical sensitivity proffered by this system, the tail profile macros are upgradeable and platform independent.
Bowden
Richard D
RD
Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA.
Buckwalter
Matthew R
MR
McBride
Jeremy F
JF
Johnson
David A
DA
Murray
Byron K
BK
O'Neill
Kim L
KL
D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage; D004260:DNA Repair; D005821:Genetic Techniques; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D012984:Software; D014407:Tumor Cells, Cultured