One of my enduring problems with intriguing preliminary data, yielded by pilot experiments, is that I tend to be overly ambitious with the subsequent ratio of data acquisition to data analysis, especially if I’m using a technique that’s new to me. For various reasons/hypotheses, related both to the variable expressivity of an inherited cancer syndrome and to therapy-induced malignancies, I decided to compare DNA damage sensitivity and repair capacity in tumor and primary cells of the peripheral nervous system. My preliminary data indicated that the ability to repair DNA damage, induced by doxorubicin treatment or gamma radiation, varied according to the genetic background of the tumor, and to the age of the animal. Two different projects (and hopefully two publications in different journals), but the same technique: single-cell gel electrophoresis, or the comet assay.

Comets, peripheral nerve sheath tumor cell line

I use the alkaline modification of the comet assay (Fairbairn et al., 1995), which allows measurement of low levels of DNA damage, including single and double strand breaks, as well as incomplete excision repair sites. When cells are lysed and subjected to electrophoresis in a thin agarose gel poured onto a microscope slide, a “comet” develops, which can be visualized on a fluorescence microscope, following propidium iodide staining. The size and shape of the comet, and distribution of DNA therein, are correlated with the amount of DNA damage. Several software packages, developed specifically for analysis of DNA comets, allow one to obtain many different parameters from images such as the ones I’ve included in this post: tail length, percentage of DNA in the tail, tail extent moment, comet profile, Olive tail moment. Olive tail moment (OTM) is a parameter often included in publications using the comet assay; it’s the product of the percentage of DNA in the tail and the distance between the intensity centroids of the head and the tail along the x-axis of the comet (Vilhar, 2004).

Comets, primary Schwann cells

So, to get an idea of whether all this data crunching would be worthwhile, I set up some preliminary experiments at several time points, and just looked for disappearance of the comet tails, which indicated that the DNA damage had been repaired. I noticed differences in the time courses of DNA damage repair for murine PNST cell lines from two different genetic backgrounds, and for Schwann cells isolated from mice at different ages and of two different genotypes, between three and twenty-one months. Based on these results (and some related experiments), I got a small amount of funding to perform more detailed comet analyses (not particularly expensive experiments, really). You can imagine what happened next … while I had the cells, and the time between busy teaching stints, I cranked out the comet assays and captured hundreds and hundreds of comet images at different DNA repair time points. The comet gel slides don’t last (at least not if you use the inexpensive homemade version), and so the images have to be captured on the day that you run the assays.

Now, my unmeasured chickens comets have come home to roost, and I can be found at the computer, analyzing comet images, in every spare moment between lectures and anatomy labs at work. I don’t even want to discuss assembling the figures and tables and manuscripts at this point.

References:

Fairbairn DW, Olive PL, O’Neill KL (1995) The comet assay: a comprehensive review. Mutation Research 339, 37-59.

Vilhar B (2004) Help! There is a comet in my computer! http://botanika.biologija.org/exp/comet/comet_guide01.pdf