Medium-throughput comet assay

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• Created: Thursday, 27 August 2015 10:29

Researchers from North-West University, South Africa have presented a methods paper describing “a medium-throughput comet assay to evaluate the global DNA methylation status of single cells”.  The comet assay is a simple and cost effective technique, commonly used to analyse and quantify DNA damage in individual cells and the versatility of the comet assay allows introduction of various modifications to the basic technique.

Here, the investigators used the difference in the methylation sensitivity of isoschizomeric restriction enzymes to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells grown in cell cultures. DNA methylation is not only important for maintaining genome stability, but also plays an important role in gene regulation. DNA methylation is an epigenetic event which involves the chemical modification of DNA wherein the DNA sequence is not changed. The adaption of the comet assay to measure global methylation relies on the isoschizomeric properties of the two restriction enzymes: MspI and HpaII.  To read more about DNA methlylation, and the use of the comet assay to measure methylation, please refer to the original publication.

In the experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to measure changes in the global DNA methylation pattern in individual cells under various growth conditions.  The medium-throughput comet assay (as described by the authors) utilised a “12-well gasket”, which was used for the preparation of the comet slides and to perform the enzymatic digestion.

Comet images were captured with an Olympus IX70 fluorescence microscope (200× magnification) and scored using Comet IV computer software version 4.3.1 (Perceptive Instruments Ltd). At least 400 comets were randomly scored per sample (between 50 and 100 comets per well) and the percentage of DNA migrating from the comet head (tail intensity) was measured for each comet scored. Nine replicates of three independent experiments were performed for each sample.

For full experimental information, and other details the medium-throughput methylation sensitive comet assay, please refer to the original publication.

The scientists concluded that the difference in methylation sensitivity of the enzymes HpaII and MspI may be exploited to demonstrate the feasibility of using the comet assay to measure global DNA methylation level in individual cells.

In this investigation, they showed that the comet assay can be modified to measure global DNA methylation in single cells in a medium-throughput manner. The scientists believe that the use of the 12-well gasket to perform the enzyme digestions offers improved conditions for enzymatic digestion and overcomes some of the limitations that are faced when restriction enzymes are used in conjunction with the comet assay, such as edge-effects, sub-optimal enzyme reaction conditions and gel drying.

The authors concluded that the comet assay remains an attractive assay to use due to its inexpensive nature, tissue specificity and ability to measure the changes in the global DNA methylation pattern of a variety of cells under different physiological conditions on a single cell level.

The authors also believe that due to the modifications made in this study, the versatility of the comet assay is increased.  Furthermore,  it was concluded in this study that an increased number of observations that can be made during a single experiment and consequently labour and inter-experimental variability are both reduced.

This case study is based upon:

Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells

Angelique Lewies, Etresia van Dyk, Johannes F. Wentzel and Pieter J. Pretorius.  Front. Genet. 5:215. 2014