The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.
Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the “scoring” of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.
Cells are exposed continually to agents arising from the internal and external environments, which can damage DNA1,2. This damage can cause aberrant cell function3, and therefore DNA damage may play a critical role in the development of many major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging4. The comet assay (also called single-cell gel electrophoresis) is an increasingly popular method for detecting and quantifying cellular DNA damage.
At its simplest, the alkaline comet assay (ACA) detects strand breaks (SB; both single and double), together with apurinic/apyrimidinic sites and alkali-labile sites (ALS) both of which become single-strand breaks under alkaline conditions5. The neutral pH comet assay can evaluate frank single- and double-strand breaks6. Furthermore, the ACA, in combination with a number of DNA repair enzymes, can detect a considerable range of types of DNA damage, e.g., oxidized purines (identified by the use of human 8-oxoguanine DNA glycosylase 1; hOGG17); oxidized pyrimidines (using Endonuclease III; EndoIII) and cyclobutane pyrimidine dimers (using T4 endonuclease V; T4endoV)8. The comet assay can also be used to evaluate DNA lesions induced by crosslinking agents, such as cisplatin9,10,11. As indicated by the assay's formal name, i.e., single cell gel electrophoresis, the assay relies upon the cells under analysis being a single cell suspension; most commonly, these are cultured cells but may be isolated from whole blood12,13, or whole blood itself can be used14,15. Alternatively, a single cell suspension may be generated from solid tissues.
Apart from a few exceptions, most notably the CometChip reports from the Engleward lab16, the overall comet assay protocol has not changed dramatically from that originally described by the assay's inventors (Östling and Johansson17 and Singh et al.18). The comet assay involves numerous steps (Figure 1). Many of these steps involve the transfer of the thin, cell-containing agarose gels, one slide at a time, and, therefore, pose a risk of damage or loss of the gel, jeopardizing the experiment's success. Consequently, the comet assay can be time-consuming, particularly if a significant number of slides are being run. Typically, a maximum of 40 slides are run in a large (33 cm x 59 cm x 9 cm) electrophoresis tank, which sits within an even larger tray containing wet ice for cooling. It has been recently reported that the assay runtime can be shortened to 1 day by decreasing the duration of the lysis step and not drying the slides before staining19.
The present authors have previously reported a novel approach to the high throughput alkaline comet assay (HTP ACA), in which multiple (batches of 25) comet assay microscope slides can be manipulated simultaneously throughout the comet assay process20,21,22. This patented approach minimizes the risk of damage to, or loss of, the sample-containing gels by removing the need to manipulate the microscope slides individually and can be applied to all variants of the comet assay, which use microscope slides. The slide-containing racks protect the gels during the manipulations, and consequently, the sample processing is quicker and more efficient. The slides can also undergo electrophoresis in the racks, held in the vertical, rather than horizontal, orientation. This, and integral cooling, significantly decrease the footprint of the electrophoresis tank and removes the need for wet ice. Taken together, this represents a significant improvement over the conventional procedure. The equipment used is illustrated in Figure 2. The protocols described here, using this novel approach, demonstrate the representative application to cultured cells and whole blood14 for detection of alkali-labile sites (ALS), DNA inter-strand crosslinks (ICL), and the substrates of various DNA repair enzymes.
This study demonstrates the versatility provided by the current equipment, which can be used to achieve high throughput with a variety of representative, common variants of the comet assay (i.e., alkaline, enzyme-modified, blood, and ICL, and other variants will be suitable too). In addition, the present approach brings with it several benefits20,21: (a) assay run time is decreased due to manipulation of multiple slides in parallel (handling time decreases by 60%); (b) risk of damage to gels, and hence the risk to the experiment are decreased; (c) reagent requirements are decreased (e.g., the volume of the electrophoresis tank is smaller than the conventional tank); (d) the number of slides run is increased. One tank can provide a 20% increase in the number of slides run compared to a single conventional tank; however, multiple electrophoresis tanks can be run or slaved (i.e., multiple tanks controlled by a single power supply), in parallel from the same power supply, and still require a benchtop footprint smaller than a single conventional tank with ice tray; and (e) tank footprint is decreased due to vertical orientation of slides and integral cooling (saves lab space); the HTP tank comprises a high-performance ceramic cooling base with a sliding drawer that can fit one frozen cooling pack to maintain optimal buffer temperature without having to perform the process in a cold room.
Moreover, the chilling plate developed by us accommodates 26 comet slides, enables rapid solidification of the low melting point agarose on the comet assay slides and facilitates an easy retrieval of the slides after the agarose gel is solidified. The above innovations make the comet assay process simpler and easier.
While other high-throughput approaches have been developed (e.g., 12-gel comet assay, CometChip, or 96 mini-gel formats)25, many scientists prefer using the conventional microscope slides (which includes the commercially available pre-coated slides, or other specialized slides). The present approach can accommodate all types of microscope slides, allowing experiments using these slides to be scaled up through faster slide processing and handling. As noted above, the HTP comet system brings many advantages, but there is one notable limitation: the current approach provides only a 20% increase in the number of samples run, compared to a conventional horizontal tank (although processing of slides is much faster). The CometChip and 96 mini-gel formats run a greater number of samples. To date, we do not know whether the present approach can accommodate the CometChip or 96 mini-gel formats, although we predict that it will. As noted above, the number of samples can be increased further by slaving tanks to a single power supply. As with all approaches, there is still a chance of losing or damaging the gels while loading samples and analyzing them under the microscope, but this is more due to operator error, and the chances of this are minimized with the current approach.
The use of the HTP comet system can greatly help analyze DNA damage, facilitating the use of the comet assay in a wide range of applications, such as molecular epidemiology, male reproductive science, genotoxicology studies, and environmental toxicology. This is particularly true for those users who wish to have all the benefits of improved throughput, and ease of use, without moving away from the familiar, cost-effective, conventional microscope slides.
The authors have nothing to disclose.
The work reported in this publication was, in part, supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under award number: 1R41ES030274. The content is solely the authors' responsibility and does not necessarily represent the official view of the National Institutes of Health.
22 x 22 mm glass coverslips | Fisher Scientific, Hampton, NH, USA | 631-0124 | |
A2780 | ECACC, Louis, MO, USA |
93112519 | |
Concentrated nitric acid (OptimaTM grade) | Fisher Scientific Fair Lawn, NJ, USA | A467-250 | |
Fluorescence microscope equipped with a camera | Zeiss, Jena, Germany | ||
Fresh human whole blood | Zen Bio Inc | SER-WB10ML | Commercial human whole blood sample |
GraphPad Prism | GraphPad Software, San Diego, California | Data analysis software | |
HTP Comet Assay system | Cleaver Scientific | COMPAC- 50 | |
Human Keratinocyte (HaCaTs) | American Type Culture Collection (ATCC), Manassas, VA, USA | Discontinued | Can be purchased from another company ADDEXBIO TECHNOLOGIES Cat# T0020001 |
Hydrogen peroxide (H2O2) 30% in water |
Fisher Scientific, Hampton, NH, USA | BP2633-500 | |
ICP-MS iCAP RQ ICP-MS system |
Thermo Scientific, Waltham, MA, USA |
IQLAAGGAAQFAQKMBIT | |
Image and Data Analysis software | Perceptive Instrument, Bury St Edmunds, England, UK |
125525 | Free image analysis softwared is available e.g., ImageJ |
Internal Standard Mix | SPEX Certiprep, Metuchen, NJ, USA |
CL-ISM1-500 | Bismuch (isotope monitored 209 Bi)-concnetration of 10 µg/mL in 5% HNO3 |
Low melting point Agarose | Invitrogen Waltham, MA, USA |
P4864 | |
Na2EDTA (disodium ethylenediaminetetraacetic acid) | Sigma Aldrich, St. Louis, MO, USA |
E5134 | |
NaCl (Sodium chloride) | Sigma Aldrich, St. Louis, MO, USA |
S7653 | |
NanoDrop One | Thermo Scientific, Waltham, MA, USA |
701-058108 | Nanodrop for measuring DNA concentration |
Nanopure Infinity Ultrapure Water System (Barnstead Nanopure) | Thermo Scientific, Waltham, MA, USA |
D11901 | Ultrapure water (16 MΩ cm-1) |
NaOH (sodium Hydroxide) | Sigma Aldrich, St. Louis, MO, USA |
E5134 | |
Normal melting point Agarose | Fisher Scientific, Hampton, NH, USA |
16520100 | For pre-coating slides |
OCI-P5X | University of Miami, Miami, FL, USA |
N/A | Live Tumor Culture Core facility provided the cells |
Platinum (Pt) reference standard | SPEX Certiprep, Metuchen, NJ, USA |
PLPT3-2Y | (1000 µg/mL in 10% HCl) containing Bismuch |
Propidium Iodide (1.0 mg/mL in water) |
Sigma Aldrich, St. Louis, MO, USA |
12-541BP486410ML | |
QIAamp DNA Mini Kit | Qiagen Valencia, CA, USA |
51304 | DNA extraction Kit |
Single-frosted glass microscope slides | Fisher Scientific, Hampton, NH, USA |
12-541B | |
SKOV3 | ECACC, Louis, MO, USA |
91091004 | |
Slide box | Fisher Scientific, Hampton, NH, USA |
03-448-2 | Light proof, to protect cells from the formation adventitious damage (according to the widely held view) and prevent fading of the fluorescent dye |
Slide Chilling plate | Cleaver Scientific, Rugby, England, UK |
CSL-CHILLPLATE | |
Treatment dish | Cleaver Scientific, Rugby, England, UK |
STAINDISH4X | |
Tris-base | Sigma Aldrich, St. Louis, MO, USA |
93362 | |
Triton X-100 | Fisher Scientific, Hampton, NH, USA |
BP151-500 | |
Trypsin EDTA (0.5%) | Invitrogen Gibco, Waltham, MA, USA |
15400054 | |
Vertical Slide Carrier | Cleaver Scientific, Rugby, England, UK |
COMPAC-25 |