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>

<ol>
	<li>van der Leede, B. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Alkaline+Comet+Assay+and+Enzyme-modified+Alkaline+Comet+Assay+for+Measuring+DNA+Strand+Breaks+and+Oxidative+DNA+Damage+in+Rat+Liver.">In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver.</a> Journal of Visualized Experiments. (111), 53833 (2016).</li><li>Hobbs, C. A., Chhabra, R. S., Recio, L., Streicker, M., 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=Genotoxicity+of+styrene-acrylonitrile+trimer+in+brain,+liver,+and+blood+cells+of+weanling+F344+rats.">Genotoxicity of styrene-acrylonitrile trimer in brain, liver, and blood cells of weanling F344 rats.</a> Environmental and Molecular Mutagenesis. 53 (3), 227-238 (2012).</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=Comprehensive+evaluation+of+the+flavonol+anti-oxidants,+alpha-glycosyl+isoquercitrin+and+isoquercitrin,+for+genotoxic+potential.">Comprehensive evaluation of the flavonol anti-oxidants, alpha-glycosyl isoquercitrin and isoquercitrin, for genotoxic potential.</a> Food and Chemical Toxicology. 113, 218-227 (2018).</li><li>Pant, K., 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=Vehicle+and+positive+control+values+from+the+in+vivo+rodent+comet+assay+and+biomonitoring+studies+using+human+lymphocytes:+historical+database+and+influence+of+technical+aspects.">Vehicle and positive control values from the in vivo rodent comet assay and biomonitoring studies using human lymphocytes: historical database and influence of technical aspects.</a> Environmental and Molecular Mutagenesis. 55 (8), 633-642 (2014).</li></ol>

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&#39;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>KH2PO4</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>Na2EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</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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JoVE Journal

Genetics

9.4K 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, ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

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 ...

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model organism. However, little is known about the sex-determination mechanisms found in other non-model hymenopteran taxa, such as ants. Because of the complex nature of the life cycles that have evolved in hymenopteran species, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory. Here, we describe the methods for conducting inbreeding crosses and for evaluating the success of those crosses in ant Vollenhovia emeryi. Inducing inbreeding in the laboratory using V. emeryi, is relatively simple because of the unique biology of the species. Specifically, this species produces androgenetic males, and female reproductives exhibit wing polymorphism, which simplifies identification of the phenotypes in genetic crosses. In addition, evaluating the success of inbreeding is straightforward as males can be produced continuously by inbreeding crosses, while normal males only appear during a well-defined reproductive season in the field. Our protocol allow for using V. emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ant species.

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

In this protocol, methods for conducting inbreeding crosses, and for evaluating the success of those crosses, are described for the ant Vollenhovia emeryi. These protocols are important for experiments aimed at understanding the genetic basis of sex determination systems in Hymenoptera.

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences.
This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard.
Following this guide, students should be able to:
&#8226;&#160; Harvest and extract DNA from buccal mucosa epithelial cells 
&#8226;&#160; Perform a PCR experiment and understand the function of various reaction components 
&#8226;&#160; Analyze the amplicons by agarose gel electrophoresis and interpret the results 
&#8226;&#160; Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Here we describe a simple protocol to generate DNA fingerprinting profiles by amplifying the VNTR locus D1S80 from epithelial cell DNA.

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a ...