Recommendations to enhance
reproducibility and reliability in Comet assay
Rashini Yasara Baragama-arachchi1,2
Dr. Jagath Weerasena1
Dr. Shiroma Handunnetti 1
Dr. Radhika Samarasekara2
1Institute
of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri
Lanka
2Industrial Technology Institute, Sri Lanka
Introduction
• Comet assay is a technique predominantly use in the
field of toxicology to assess the DNA damages in
single cell suspensions (Nandhakumar et al, 2011)
• Also known as Single cell gel electrophoresis assay
(SCGE assay)
• The concept of Single cell gel electrophoresis assay
was first introduced by Ostling and Johanson in 1984
• Later it was developed by N.P. Singh in 1988
Advantages
• It can be applied both in vitro and in vivo to
virtually any cell type or cell line from prokaryotic
and eukaryotic organisms
• Require only small numbers of cells per sample
(<10,000)
• Sensitivity for detecting low levels of DNA damage
• Does not require cumbersome techniques like
radiolabelling
• Low cost
• Flexible
• Rapid
Applications
1. Genetic toxicology
For screening and regulatory testing of industrial
chemicals, pharmaceuticals, biocides, cosmetics
and various herbal extracts use for different
purposes.
2. Human biomonitoring
Occupational exposure to hazardous chemicals,
pollutants and radiation
3. Ecogenotoxicology
Monitoring contamination of the environment
by genotoxic agents
Applications
4. Mechanistic studies
DNA damage & repair
5. Nutrition
Harmful and beneficial effects of diet and dietary components
6. Clinical
Diagnosis of disease and monitoring effects of treatment
7. Molecular epidemiology
Assessing inter-individual differences in susceptibility to DNA
damage and capacity to repair
Principle
Alkaline unwinding
http://www.rndsystems.com
Electrophoresis
Justification
• Method of choice for evaluation of potential genotoxicity of
various chemicals and prospective therapeutics
• Accepted as a part of battery of assays used for regulatory
submissions in genetic toxicology by regulatory authorities
(Collins et al, 2008)
• Yet the major drawback of this technique is the unreliability
in making reproducible data, due to miscellaneous
conditions used in different laboratories and due to lack of
understanding of the critical steps (Azqueta et al, 2011)
Objectives of the study
To optimize the Comet assay to enhance
reproducibility and reliability
Preparation of cells for comet assay
Cell culture
• Human lymphocytes were extracted from whole blood obtained from healthy
volunteers
• Ethical approval was obtained
• Initial viable cell count was determined by performing Trypan blue dye
exclusion assay
• cells were seeded in 6-well plates at 2x 10 5cells/well
• Lymphocytes were incubated for 1 hour at 37ºC with hydrogen peroxide
(H2O2, 200 µM) as positive control and 1xPBS as negative control
• Cell viability after the treatment was evaluated by performing Trypan blue dye
exclusion assay
Preparation of base slides
Hot Normal melting
agarose (NMA)
Absolute methanol
Store at RT
Preparation of micro-gel slides
a) Preparation of 0.5% Low melting agarose (LMPA)
• The required amount of LMPA was made freshly during
the day of the assay without microwaving
• Instead the tube containing the LMPA and PBS was
placed in a boiling water bath until LMPA dissolved and
placed in a 37 °C water bath for 20 min before use
Preparation of micro-gel slides
b) Embedding cells in LMPA and coating of base slides
90 µl
100 µl
Cell suspension
(80 µl )
2nd agarose coat
Refrigerated for 30 min
90 µl
3 rd agarose coat
LMPA at 37 °C
Refrigerated for 30 min
Remove coverslip
Cell lysis and Alkaline unwinding of DNA
Cell lysis
• Cells were lysed for 2 h at 4 ˚C
• Slides were gently washed with chilled distilled water to remove
traces of detergent
Alkaline unwinding of DNA
• Slides were placed on the middle of the platform
in an electrophoresis tank
• Slides were covered with chilled electrophoresis
buffer
• Incubated for 30 min to allow for unwinding of
the
DNA and to expose of ALS
Poland and McLeish, 2008
Electrophoresis
• Electrophoresed at 17 V and 164 mA for 45 min at 4 °C
• A software developed by Gunnar Brunborg from National
Institute of Public Health, N-0462 Oslo, Norway was used to
calculate the accurate voltage for electrophoresis (Collins et al;
2008)
Spreadsheet to calculate voltages and currents in an electrophoresis
tank.docx
Neutralization and visualization
Neutralization and fixing of slides
•
Slides were dipped in cold neutralization buffer, air dry and fixed
with absolute methanol
Staining & visualization of slides
• Slides were stained with 45 µl of Ethidium bromide [EtBr] (20 µg/
ml), left for 5 min and then dipped in chilled distilled water to
remove excess stain
• Visualized under 40x objective of the fluorescent microscope
Comet scoring and statistical analysis
• 100 cells per slide were assessed.
• “Casp 1.2.3b.1” image analysis software was used to assess the
quantitative and qualitative extent of DNA damage in the cell
• Results were analyzed using SPSS statistical software (version17.0)
• The results were considered to be significantly different at P < 0.05
Measured parameters
Parameter
Definition
Percentage of
DNA in the
tail
Fraction of DNA in the tail as
compared to the whole image
(Albertini et al, 2000)
Tail moment
(TM)
Tail length X Fraction of DNA in the
tail (Lovell et al, 2008)
http://www.cellbiolabs.com
Cell viability after treatment
Percentage (%)
100
Cell viability
95
90
85
80
75
Negative control
Positive control
• Cell viability was >80 % after treatments
Optimized conditions
Optimization of conditions for comet assay to achieve
reproducible and reliable data
1.
2.
3.
4.
5.
Optimization of conditions for preparation of base slides
LMPA preparation method
Solidification times of 2nd & 3rd agarose layers
Lysis duration
Electrophoresis conditions
– Voltage
– Duration
Optimization of conditions for preparation of base slides
Agarose concentration
Repeated heating of agarose
Alteration of actual concentration of
agarose (1%)
No significant effect on the overall process
LMPA preparation method
Method 01
Method 02
Prepared in bulk
and re-melted by
microwaving
No migration of DNA
after
electrophoresis
Prepared freshly
on the day of
assay using a
boiling water
bath
Proper migration of
DNA after
electrophoresis
Solidification times of 2nd & 3rd agarose layers
Solidification
times
10 min
20 min
Detachment of agarose
layers when removing
the cover slips
30 min
Adequately
solidified
agarose layers
Lysis duration
1 hour
Insuficiently lysed
2 hours
Fully lysed cells
Overnight
Cells were lysed and
DNA dispersed
Electrophoresis conditions
• Alkaline unwinding
Optimal unwinding time was found to be 30 min
• Electrophoresis
Parameter
Optimized condition
Voltage
24V, 17 V
Duration
30, 45, 60 min
Comet formation
Positive Control (C+)
200 µM H2O2
Negative Control (C-)
PBS
Genotoxic potential of H2O2
*
120
100
TM
80
60
• Negative control – Vehicle
(PBS)
40
20
• Positive control – 200 µM H2O2
0
Negative control
45
40
35
30
25
20
15
10
5
0
Positive control
*
% Tail DNA
* p < 0.05 when compared to
Negative control
Negative control
Positive control
Mean values of TM, OTM and Tail DNA percentage of Comets (n=100); Error bars indicate: Mean ± SEM
Conclusions
• Concentration of NMA does not affect final outcome as it
only provide a better anchorage for subsequent agarose
layers
• The most critical parameters are
1.
Concentration of LMPA, as DNA migrate through LMPA
2.
Electrophoresis voltage. It MUST be 1V/cm, not 24 V
• Comet assay was a very sensitive technique
Sensitivity means here is not that its ability to detect low level of DNA
damages, but the extreme care that has to be taken when performing
each and every step to achieve better reproducible results
References
1.
2.
3.
4.
5.
6.
Albertini RJ, Anderson D, Douglas GR, Hagmar L, Hemminki K, Merlo F et al.
IPCS guidelines for the monitoring of genotoxic effects of carcinogens in
humans. (2000) Mutation Research :463 ;111–172
Azqueta A, Gutzkow KB, Brunborg G and Collins AR. Towards a more reliable
comet assay: Optimising agarose concentration, unwinding time and
electrophoresis conditions (2011) Mutation Research: 724; 41-45
Collins AR. The comet assay for DNA damage and repair: principles,
applications, and limitations. (2004b) Molecular Biotechnology: 26(3); 249-61
Collins AR, Oscozi AA,Brunborg G, Gaiva I, Giovannelli L, Kruszewski M et al.
REVIEW:The comet assay: topical issues. (2008) Mutagenesis: 23 (3 ) ;143–151
Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P et
al. Recommendations for conducting the in vivo alkaline Comet assay. (2003)
Mutagenesis:18(1); 45–51
Morley N, Rapp A, Dittmar H, Salter L, Gould D, Greulich KO et al. UVA-induced
apoptosis studied by the new apo/necro-Comet-assay which distinguishes
viable, apoptotic and necrotic cells. (2006) Mutagenesis: 21( 2 ); 105–114
References
7.
8.
9.
Nandhakumar S, Parasuraman S, Shanmugam M, Rao KR, Chand
P and BhatBV. Evaluation of DNA damage using single-cell gel electrophoresis
(Comet Assay). (2011) Journal of Pharmacology and Pharmacotherapeutics:
2(2); 107–111
Singh N, Lai H. 60 Hz magnetic field exposure induces DNA crosslinks in rat
brain cells. (1998) Mutation Research: 400(1-2); 313-20
Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H et al.
Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic
toxicology testing. (2000) Environmental and Molecular Mutagenesis: 35;
206-221
Acknowledgement
National Science Foundation of Sri Lanka