The authors are indebted to Lincoln Martin and Kelley Owens for expert technical assistance preparing and scoring comet slides and Dr. Carol Swartz for performing statistical analyses. The authors also acknowledge the supportive contributions of members of the Genetic Toxicology, Investigative Toxicology, and Necropsy programs at ILS.

Tissues were harvested during the conduct of studies performed at AAALAC-accredited facilities at NTP contract laboratories in accordance with Good Laboratory Practice regulations (21 CFR Part 58) and animal use protocols approved by the Institutional Animal Care and Use Committee (IACUC) at each laboratory.
1. Tissue Harvest and Processing
NOTE: It is useful to prepare duplicate sample tubes (e.g., liver) or transfer approximately half of a sample to another storage tube (e.g., duodenum, stomach) to enable reanalysis, if necessary. To minimize potential sample-to-sample variability for intestinal tract tissues, it is recommended that care be taken to sample the same region of tissue relative to the stomach for each animal, and a sample be divided to generate duplicate samples. Plastic forceps are recommended for transferring sticky tissues such as brain. As an option, minced, scraped or homogenized tissue preparations may be filtered to achieve homogeneous single cell suspensions using a 40 &#181;m cell strainer attached to a conical tube.
<ol>
	<li>Remove any attached connecting tissue, organs, and debris from organ(s) of interest. Immediately following removal, swish the organ vigorously in a medium-sized weigh boat containing ~7 mL of cold freshly prepared mincing solution (Mg++, Ca++ and phenol red-free Hank&#8217;s Balanced Salt Solution, 10% v/v DMSO, and 20 mM EDTA pH 7.5) to remove residual blood and debris.</li>
	<li>Transfer each organ to another clean weigh boat containing sufficient (~7 mL) cold mincing solution to maintain the tissue submerged, on ice, until further processing.</li>
	<li>Remove a section of each organ of interest and place it in an embedding cassette. Fix in 10% neutral buffered formalin, trim, and paraffin embed according to the laboratory&#8217;s standard procedures for possible future histopathology evaluation.</li>
	<li>For the liver, cut a 5 mm longitudinal section of the left lobe and gently swish in ~7 mL of cold mincing solution. Place the strip of tissue into a clean medium-sized weigh boat containing ~7 mL of cold mincing solution and maintain on ice until ready to process further (step 1.8 or 1.11).</li>
	<li>For the duodenum, cut a 10 mm portion of the duodenum proximal to the stomach. Using a 21&#8211;25 G needle, flush to remove debris and bacteria.&#160;
	<ol>
		<li>Insert needle into one end of the duodenum and flush with 1 mL of cold mincing solution.</li>
		<li>Flush the other direction by flipping the duodenum over and repeating with another 1 mL of mincing solution. Discard the needle.</li>
		<li>Slice the duodenum open and rinse it in ~7 mL of mincing solution before putting it into a medium-sized weigh boat containing ~7 mL of clean mincing solution; maintain on ice until ready to process further (step 1.8 or 1.11).</li>
	</ol>
	</li>
	<li>For the brain, it may be useful to first divide the brain into two hemispheres; one hemisphere can be saved for possible histopathology (see step 1.2).
	<ol>
		<li>Dissect the brain region(s) of interest and gently swish in ~7 mL of cold mincing solution.</li>
		<li>Transfer tissue(s) to a clean medium-sized weigh boat containing ~7 mL of cold mincing solution and maintain on ice until ready to process further (step 1.8 or 1.11; note that small regions such as the cerebellum and hippocampus may not require any further trimming).</li>
	</ol>
	</li>
	<li>For the stomach
	<ol>
		<li>Remove and discard the forestomach. Cut open the glandular stomach and wash free from food using ~7 mL of cold mincing buffer in a medium-sized weigh boat.</li>
		<li>Remove a 5 mm strip of glandular stomach proximal to the duodenum for fixation for possible histopathology evaluation (see step 1.2).</li>
		<li>Place the remaining stomach into ~7 mL of cold mincing buffer in a medium-sized weigh boat and incubate on ice for 15&#8211;30 min. 
		NOTE: This incubation step may not be necessary; this step was included in the JaCVAM validation protocol, but is not mentioned in the OECD test guideline8,9.</li>
		<li>Transfer the stomach to a clean piece of paraffin (or Petri dish or weigh boat) and gently scrape the surface epithelium two or more times using a scalpel blade or a polytetrafluoroethene (PTFE) scraper to remove debris. Pick up the gastric mucosa with forceps and rinse with 1 mL of cold mincing buffer using a pipet. Transfer the stomach tissue to a clean surface or dish.</li>
		<li>Carefully scrape the stomach epithelium 4&#8211;5 times (more, if necessary) in mincing solution (typically 250 &#181;L/mouse or 500-1,000 &#181;L/rat) with the back of a scalpel blade or PTFE scraper to release the cells. Optionally, rinse tissue with an approximately equal volume of mincing solution to recover more cells. Collect the mincing solution containing released cells using a pipet and transfer to a microcentrifuge tube on ice.</li>
	</ol>
	</li>
	<li>For mincing tissue samples, cut a 3&#8211;4 mm section of liver or brain tissue or ~5 mm of duodenum and transfer to a labeled 1.5 or 2.0 mL microcentrifuge tube containing 1 mL of cold mincing solution. Rapidly mince (&#8804;30 s) with mincing scissors until finely dispersed, while keeping the sample cold. The sample should look slightly cloudy; however, it is common for some pieces to remain that will settle to the bottom of the tube.</li>
	<li>To use the tissue fresh, maintain tubes containing minced tissue or scraped epithelial cells on ice; use the tissue to prepare comet slides within approximately 1 h of harvest (outlined in section 2).</li>
	<li>For freezing the tissue samples, immediately following mincing or collection of scraped epithelial cells
	<ol>
		<li>Secure the tube lid and drop tube into a Dewar flask containing liquid nitrogen; tubes may be maintained in liquid nitrogen for the duration of necropsy (&#8804;3 h).</li>
		<li>Retrieve tubes using a ladle and place on dry ice to sort. Transfer tubes to a freezer box on dry ice and store box in a -80 &#176;C freezer.</li>
	</ol>
	</li>
	<li>For preparing frozen cubes of tissue, cut several small pieces of tissue (&#8804;4 mm in diameter and ~6 mm in length; Figure 1).
	<ol>
		<li>Individually drop them directly into a medium-sized weigh boat or other suitable vessel containing liquid nitrogen.</li>
		<li>Wait a few seconds for the pieces to freeze completely and transfer individual frozen tissue cubes to a labeled microcentrifuge tube or cryotube maintained on dry ice; do not allow the pieces to clump together during the freezing process.</li>
		<li>Transfer tubes to a freezer box on dry ice and store box in a -80 &#176;C freezer.</li>
	</ol>
	</li>
</ol>
2. Slide Preparation
<ol>
	<li>For minced/scraped tissue
	<ol>
		<li>Maintain tubes containing fresh tissues on wet ice.</li>
		<li>Place tubes of frozen tissue into a room temperature water bath until the samples are completely thawed, while ensuring they are kept cold. Immediately transfer the tubes onto ice.</li>
		<li>Immediately prior to withdrawing cells for transfer to agarose (step 2.3), mix each cell suspension gently; for non-filtered samples, allow large chunks to settle to the bottom of the tube. Maintain all tubes on ice until slides have been prepared for all samples.</li>
	</ol>
	</li>
	<li>For cubed tissue
	<ol>
		<li>Retrieve tubes containing tissue cubes from the 80 &#176;C freezer and maintain on dry ice until slide preparation.</li>
		<li>Aliquot 1 mL of Merchant&#8217;s medium (0.14 M NaCl, 1.47 mM KH2PO4, 2.7 mM KCl, 8.1 mM Na2HPO4, 10 mM NaEDTA, pH 7.4) or mincing solution into a labeled 1.7 mL microcentrifuge tube for each sample and maintain on ice.</li>
		<li>Working rapidly to avoid tissue thawing, place a cube of tissue into the open end of a room temperature tissue mincing device (Figure 1). Multiple tissue pieces may be used; if necessary, the frozen tissue may be cut or cracked into smaller pieces using a cold mortar and pestle to reduce the cube size to fit into the device.</li>
		<li>Quickly insert a size-matched syringe plunger into the device to push the tissue to the mesh end which should then be placed into the microcentrifuge tube containing cold mincing solution; avoid forcing the liquid to overflow the tube. Immerse the mesh end of the device for approximately 5&#8211;10 s in the cold mincing solution to allow the tissue to thaw completely.</li>
		<li>Completely depress the plunger until cells are seen extruding through the mesh pores. Then pull the plunger up approximately 2.5 cm and press down again completely; repeat the process several times until the mincing solution becomes turbid.</li>
		<li>If necessary, the sample may be concentrated to increase cell density by refrigerated centrifugation for 5 min at 1100 rpm and removal of a portion of the supernatant.</li>
		<li>Repeat the process for all samples using a clean tissue mincing device for each sample.</li>
	</ol>
	</li>
	<li>Transfer 50 &#181;L of cell suspension to a tube containing 450 &#181;L of 0.5% low melting point agarose at 37 &#176;C. 
	NOTE: Volumes may be adjusted, but the amount of agarose should be &#8804;1%.</li>
	<li>Prepare and score slides as described previously10,11.</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+and+data+interpretation+of+the+in+vivo+comet+assay+in+pharmaceutical+industry:+EFPIA+survey+results.">Performance and data interpretation of the in vivo comet assay in pharmaceutical industry: EFPIA survey results.</a> Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 775-776, 81-88 (2014).</li><li>ICH. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Conference+on+Harmonisation+of+Technical+Requirements+for+Registration+of+Pharmaceuticals+for+Human+Use+(ICH)+S2(R1):+Guidance+on+Genotoxicity+Testing+and+Data+Interpretation+for+Pharmaceuticals+Intended+for+Human+Use.">International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use.</a> ICH. , (2012).</li><li>EFSA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientific+opinion+on+genotoxicity+testing+strategies+applicable+to+food+and+feed+safety+assessment.">Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment.</a> EFSA Journal. 9 (9), 2379 (2011).</li><li>Lorenzo, Y., Costa, S., Collins, A. R., Azqueta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay,+DNA+damage,+DNA+repair+and+cytotoxicity:+hedgehogs+are+not+always+dead.">The comet assay, DNA damage, DNA repair and cytotoxicity: hedgehogs are not always dead.</a> Mutagenesis. 28 (4), 427-432 (2013).</li><li>Guerard, M., Marchand, C., Plappert-Helbig, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+experimental+conditions+on+data+variability+in+the+liver+comet+assay.">Influence of experimental conditions on data variability in the liver comet assay.</a> Environmental and Molecular Mutagenesis. 55 (2), 114-121 (2014).</li><li>Jackson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+freezing+tissues+and+cells+for+analysis+of+DNA+strand+break+levels+by+comet+assay.">Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay.</a> Mutagenesis. 28 (6), 699-707 (2013).</li><li>Recio, L., Kissling, G. E., Hobbs, C. A., Witt, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Comet+assay+dose-response+for+ethyl+methanesulfonate+using+freshly+prepared+versus+cryopreserved+tissues.">Comparison of Comet assay dose-response for ethyl methanesulfonate using freshly prepared versus cryopreserved tissues.</a> Environmental and Molecular Mutagenesis. 53 (2), 101-113 (2012).</li><li>Uno, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JaCVAM-organized+international+validation+study+of+the+in+vivo+rodent+alkaline+comet+assay+for+detection+of+genotoxic+carcinogens:+II.+Summary+of+definitive+validation+study+results.">JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for detection of genotoxic carcinogens: II. Summary of definitive validation study results.</a> Mutation Research/Genetetic Toxicology and Environmental Mutagenesis. 786-788, 45-76 (2015).</li><li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OECD+Guideline+for+the+Testing+of+Chemicals:+In+Vivo+Mammalian+Alkaline+Comet+Assay.">OECD Guideline for the Testing of Chemicals: In Vivo Mammalian Alkaline Comet Assay.</a> OECD. 489, (2016).</li><li>Hobbs, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comet+assay+evaluation+of+six+chemicals+of+known+genotoxic+potential+in+rats.">Comet assay evaluation of six chemicals of known genotoxic potential in rats.</a> Mutation Research/Genetetic Toxicology and Environmental Mutagenesis. 786-788, 172-181 (2015).</li><li>Ding, W., Bishop, M. E., Lyn-Cook, L. E., Davis, K. J., Manjanatha, M. 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This work was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (NIEHS) under contract HHSN273201300009C; however, the statements, opinions, or conclusions contained therein do not necessarily represent the statements, opinions, or conclusions of NIEHS, NIH, or the United States government.

As demonstrated previously7,12,13, properly handled flash frozen minced tissue provides good results in the comet assay. In fact, baseline % tail DNA values for frozen minced rat and mouse liver prepared in our laboratory are typically &#8804;6%, as recommended by the OECD test guideline9 for freshly minced rat liver samples. Good results have been obtained by multiple laboratories using a variety of tiss...

The comet assay is increasingly used as a means to evaluate DNA damage in cultured cells and tissues exposed to chemicals or other environmental stressors1. The assay can detect DNA double- and single-strand breaks, alkali-labile lesions, and single-strand breaks associated with incomplete DNA repair. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline for pharmaceutical testing recommends a DNA strand breakage assay such as the comet assay as a second test to supplement the rodent erythrocyte micronucleus assay for assessing in vivo genotoxicity and as a follow-up test for assessing mode of action in target organs of tumor induction2. The European Food Safety Authority (EFSA) recommends the in vivo comet assay as a suitable follow-up test for investigating the relevance of a positive result in an in vitro genotoxicity test3. In 2014, an OECD test guideline was approved for the rodent comet assay, thus increasing the acceptability of the assay for use in regulatory testing of genotoxic potential. The assay is based on the electrophoretic separation of relaxed DNA loops and fragments that migrate from nucleoids of lysed cells. Basically, single cells are embedded in agarose that has been layered onto microscope slides. Slides are then immersed in lysis buffer followed by an alkaline (pH &#62; 13) solution, which allows the tightly coiled nuclear DNA to relax and unwind. Slides are then placed in an electric field, which stimulates migration of negatively charged DNA toward the anode, creating images that resemble comets; the relative amount of DNA in the comet tail compared with the comet head is directly proportional to the amount of DNA damage; DNA content in the tail is typically quantified using digital imaging software.
Because the comet assay detects fragmented DNA, accurate quantification of exposure-induced DNA damage can be confounded by chromatin fragmentation associated with necrosis or apoptosis resulting from treatment-induced cytotoxicity or stress. Furthermore, DNA damage can occur as a result of mechanical shearing or improper sample processing4. The importance of maintaining harvested tissue chilled prior to slide preparation to minimize the baseline level of DNA damage has been previously demonstrated5,6. Many laboratories prepare comet assay slides from fresh tissue; however, this can be logistically challenging when preparing slides from multiple tissue types per animal in a study with a large number of animals. Moreover, this presents a problem when slide preparation and analysis are to occur at a remote laboratory, necessitating shipment of samples. For example, the U.S. National Toxicology Program includes the comet assay as a component of its genetic toxicology testing program (https://ntp.niehs.nih.gov/testing/types/genetic/index.html) and sometimes incorporates the assay into 28&#160;or 90 day repeat dose toxicity studies; this necessitates collection of tissue by the in-life laboratory and transfer of samples to another laboratory for analysis. To accomplish this, tissue pieces are minced, and/or epithelial cells of the gastrointestinal tract are scraped and cellular suspensions are flash frozen and stored in a freezer for subsequent shipment and storage by the receiving laboratory until analysis7. Proper handling of samples is crucial for obtaining high quality data using frozen tissue; however, reproducible manipulation of tissue samples during necropsies performed by ever-changing personnel is difficult to control, especially at in-life laboratories that do not routinely harvest tissues for the comet assay. Refresher training of necropsy staff or use of a mobile unit staffed by experienced laboratory personnel to collect fresh or frozen tissue samples is often too costly, not feasible, or simply undervalued.
To better ensure consistent generation of high quality tissue samples for transfer to a remote site for comet assay analysis, the utility of a published method6 of tissue preservation from flash frozen cubes of tissue was explored. In this method, frozen cubes of tissue are loaded into a stainless steel tissue mincing device (Figure 1) that is placed into a microcentrifuge tube containing cold buffer. The cube of tissue is then pushed through a small gauge mesh at the end of the device. Repeatedly forcing the tissue suspension through the mesh sieve in both directions several times results in a relatively uniform single cell suspension. Samples prepared by this method compared favorably in quality to both fresh and frozen tissue samples prepared by mincing. As an added benefit, unlike minced samples, tissue cubes can be stored frozen for prolonged periods of time and still yield high quality results in the comet assay.

<table><tbody><tr><td>Cryovials</td><td>Corning Costar</td><td>430488</td><td></td></tr><tr><td>Dental Wax Sheets</td><td>Electron Microscopy Sciences</td><td>72670</td><td></td></tr><tr><td>Dissecting (Mincing) Micro Scissors</td><td>Fisher Scientific</td><td>08-953-1B</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Gibco</td><td>14175-079</td><td></td></tr><tr><td>KCl</td><td>Teknova</td><td>P0315-10</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P9791</td><td></td></tr><tr><td>Low Melting Point Agarose</td><td>Lonza</td><td>50081</td><td></td></tr><tr><td>Microfuge Tubes (1.7 mL)</td><td>Corning</td><td>3207</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S7907</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S6191</td><td></td></tr><tr><td>Neutral Buffered Formalin</td><td>Leica</td><td>600</td><td></td></tr><tr><td>Scalpel Blades</td><td>Miltex</td><td>4-110</td><td></td></tr><tr><td>Syringe Plunger (1 mL)</td><td>Fisher Scientific or Vitality Medical</td><td>14-826-88; 8881901014</td><td>Becton Dickinson or Monoject tuberculin syringe</td></tr><tr><td>Tissue Mincing Device</td><td>NorGenoTech (Oslo, Norway)</td><td>None</td><td>Small variability in diameter observed which can affect snuggness of plunger.</td></tr><tr><td>Tweezers, plastic</td><td>Trade Winds Direct</td><td>DF8088N</td><td>Reinforced nylon, nonsterile, blunt tip, autoclavable; tradewindsdirect.com</td></tr><tr><td>Weigh Boats</td><td>Krackler Scientific/Heathrow Scientific</td><td>6290-14251B</td><td></td></tr></tbody></table>

use of frozen tissue in the comet assay for the evaluation of dna damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

Study 1 
Liver was harvested from two cohorts of male Sprague Dawley rats administered corn oil for 4 days, staggered by one week. Slides were prepared from freshly minced tissue, frozen minced tissue, and frozen cubed tissue processed in Merchant&#8217;s medium or mincing solution using the tissue mincing device. Frozen tissues obtained from animals from the first cohort were evaluated after freezer storage for ~3.5 months. Frozen tissues obtained from animals from the second cohort were evaluated ...

Watch this Scientific Journal Video about Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage at JoVE.com

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

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9.5K Views.  Toxicology Program, ILS, Inc.. This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

Video: Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Cancer Research

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Bioengineering

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Neuroscience

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Biology

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

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.

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 ...

Comet Assay for DNA Damage Detection in Cells

Source: Ji, Y., et al. <a href="https://app.jove.com/v/63559">A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage.</a> J. Vis. Exp. (2022).
The video demonstrates the comet assay for detecting DNA damage following exposure to harmful agents. Under alkaline conditions, the DNA unwinds and denatures, and upon electrophoresis, the undamaged and damaged DNA migrate differently in the agarose gel, forming a comet-shaped structure. The slowly migrating undamaged DNA forms the circular comet head, while the rapidly migrating damaged DNA fragments form the elongated comet tail.

Source: Ji, Y., et al. A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage. J. Vis. Exp. (2022).
The video demonstrates the comet ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

Summary

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Evaluating In Vitro DNA Damage Using Comet Assay

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Methodology for Accurate Detection of Mitochondrial DNA Methylation

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Comet Assay for DNA Damage Detection in Cells