LB8
Introducing a true internal standard for the Comet assay
Murizal Zainol2, Julia Stoute1, Gabriela Almeida3, Alexander Rapp4, Karen Bowman1, George Don Jones1
1University of Leicester, UK; 2Institute for Medical Research, Kuala Lumpur, Malaysia; 3IPATIMUP, Porto, Portugal; 4TU Darmstadt, Germany
Background
The Comet assay, a sensitive method for assessing DNA damage and repair at
the level of individual cells, is being increasingly exploited in many areas of
cancer research including basic, clinical, translational, epidemiological, and
prevention. However, many features of the assay contribute to the noted
intra-assay variability and inter-assay reproducibility. To minimise these, we
are developing internal standard materials consisting of reference cells
which have their DNA substituted with BrdU.
Method
Using a fluorescent anti-BrdU antibody, plus an additional barrier
filter, comets derived from these reference cells can be readily distinguished
from the test cell comets, present in the same gel, on analysis. In
experiments to evaluate the reference cell comets as external and internal
standards, the reference and test cells were either present in separate gels on
the same slide or mixed together in the same gel, respectively, before their
co-exposure to X-irradiation.
Results
Using the reference cell comets as internal standards led to substantial
(>2-fold) reductions in the coefficient of variation (CoV) for intra and
inter experimental measures of radiation-induced comet formation and DNA damage
repair; only minor reductions in CoV were noted when the reference and test
cell comets were in separate gels.
Conclusion
These studies indicate that differences between individual gels
significantly contribute to assay variation; having both the reference and test
cells together in the same gel reduces variation in comet measures caused by
protocol inconsistencies. Ultimately, we anticipate that further development
will deliver off the shelf QA materials for the Comet assay.
