Name: HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2’-deoxyguanosine, in Cultured Cells and Animal Tissues (Video) | JoVE Journal
Uploaded: 2015-08-01T14:00:00.000+00:00
Duration: 14 min 12 s
Description: Learn to quantify DNA oxidation using HPLC-ED to detect 8-oxo-dGuo in cultured cells and animal tissues. This method minimizes artifactual oxidation during sample preparation for reliable biomarker assessment.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This research was funded by the Health Canada Genomics Research and Development Initiative (GRDI) and the Canadian Regulatory Strategy for Biotechnology (CRSB). The authors have no conflict of interests.

Ensure that all animal husbandry, housing, handling and experimentation adhere to local rules and regulations and that experimentation protocols are approved prior to commencing any study. For the described experiments, animal care, handling, and treatment were approved by the Health Canada Animal Care Committee. See the &#8220;Reagents table&#8221; for the suppliers&#8217; information.
1. Collecting Biological Samples
<ol>
	<li>Cells or Animal Tissues
	<ol>
		<li>Grow human alveolar adenocarcinoma A549 cells in F12-K media containing 10% fetal bovine serum, 100 units/ml of penicillin and 100 &#181;g/ml of streptomycin.</li>
		<li>Seed cells at approximately 1 million cells per a 10-cm plate. For each experiment, use a set of 12 plates in total (three plates per one biological replicate x four doses) to provide enough DNA (80 &#956;g) for enzymatic digestion and HPLC analysis.</li>
		<li>When cell density becomes greater than approximately 70% of the plate surface area, remove media and wash cells twice with 4 ml of phosphate-buffered saline (PBS), pH 7.4.</li>
		<li>For cultured animal cells, use KBrO3 as a positive control. 
		NOTE: A positive linear relationship between KBrO3 concentration and 8-oxo-dGuo frequency has been reported in the literature9.</li>
		<li>Dissolve KBrO3 in PBS. Ensure that the final concentration in the cell culture medium is greater than 1 mM to obtain a statistically significant increase in 8-oxo-dGuo relative to un-exposed cells. Add the same concentration of KBrO3 to each plate in the series (e.g., 12 10-cm plates).</li>
		<li>Incubate the plates for 3 hr at 37 &#176;C. Remove the media and wash once with PBS.</li>
		<li>Add 1 ml of trypsin solution (stock concentration 2.5 g/ml) and incubate for 3 min at 37 &#176;C.</li>
		<li>Wash with 4 ml PBS, collect the cells from each set into a single 50 ml conical polystyrene centrifuge tube, and centrifuge at 1,000 x g for 5 min at 4 &#176;C.</li>
		<li>Remove PBS and store the cell pellet at -80 &#176;C until further analysis. Artificial DNA oxidation can be further minimized by nuclear isolation, as described elsewhere10.</li>
	</ol>
	</li>
	<li>Animal Tissues
	<ol>
		<li>Dose animals as required. For this protocol, adult (9 weeks of age) treat male Muta Mouse by oral gavage daily for three consecutive days with 20 mg DBC/kg bw per day dissolved in olive oil. 
		NOTE: This transgenic animal harbors engineered &#955;-bacteriophage and mutation reporter gene lacZ from E. coli11 is used for transgenic rodent mutation assays12.</li>
		<li>Perform animal necropsy e.g., anesthetized the mice can using isoflurane and then euthanized via cervical dislocation followed by chest cavity opening. Immediately flash freeze tissues in liquid nitrogen. Store tissues at -80 &#176;C until analysis. &#160; 
		NOTE: The timing of euthanasia after the treatment may be another important variable (for instance, DNA oxidation was maximal at 72 hr after noise exposure in the rat brain and liver13).</li>
	</ol>
	</li>
</ol>
2. DNA Extraction, Precipitation and Wash (For Tissues Proceed Directly to 2.2)
<ol>
	<li>Homogenize the collected cells in a 50 ml conical polystyrene centrifuge tube with 1 ml of DNA isolating agent such as DNAzol. Use a 1,000 &#956;l pipette tip to disperse the cell pellet until the solution is homogeneous. Transfer homogenate to a new 1.5 ml microcentrifuge tube. Incubate on ice for 10-20 min. Proceed to 2.3.</li>
	<li>Homogenize 15-20 mg of tissue in a 1 ml handheld nonstick glass homogenizer containing 500 &#956;l DNA isolating agent. Gently raise and lower homogenizer for about 1 min; softer tissues will require less time. Gentle treatment ensures less shearing of DNA. Store the homogenate on ice for about 10-20 min.</li>
	<li>Pellet the homogenate by centrifugation for 10 min at 10,000 x g and 4 &#176;C in a 1.5 ml conical microcentrifuge tube.</li>
	<li>Carefully transfer the resulting supernatant into a new 1.5 ml microcentrifuge tube paying careful attention to avoid contacting the pellet.</li>
	<li>Precipitate DNA from the homogenate by adding 0.5 ml 100% EtOH per 1 ml of homogenate. Invert tubes 10 times to ensure isolating reagent and EtOH are sufficiently mixed.</li>
	<li>At this point, DNA precipitate is viscous, spool it onto a plastic pipette tip (e.g., with a maximum holding capacity of 200 &#956;l) and then transferred to a new 1.5 ml conical microcentrifuge tube. &#160; 
	NOTE: Quick centrifugation can be employed to remove any remaining lysate from the isolated DNA. 
	(DNA Wash and Solubilization)</li>
	<li>Add 1 ml of 75% EtOH to the isolated DNA. Suspend the DNA pellet thoroughly by inverting the tubes 10 times.</li>
	<li>Carefully decant the ethanol from the tube. Ensure that the DNA pellet sticks to the side of the tube. Store the tubes vertically for 1-2 min and use a pipette to remove any excess EtOH from the bottom of the tube.</li>
	<li>Repeat the DNA wash once more, and either store the sample in EtOH at -20 &#176;C (stable for several months) or immediately proceed to digestion.</li>
	<li>Dissolve DNA in digestion buffer (described below) and then quantify using standard spectroscopic methods (i.e., absorbance at 260 nm using NanoDrop spectrophotometer).</li>
</ol>
3. Enzymatic Digestion
<ol>
	<li>Prepare &#8220;digestion buffer&#8221; by combining appropriate volumes (depending on the number of samples to be digested) of 50 mM monobasic Na phosphate (containing 2.0 mM KCl, 1.0 mM desferal (DFO) and 200 mM MgCl2) with 50 mM dibasic Na phosphate (also containing 2.0 mM KCl, 1.0 mM DFO and 200 mM MgCl2). Each sample requires 4.0 &#956;l monobasic and 17.0 &#956;l dibasic phosphate solution.</li>
	<li>Remove any remaining EtOH from the bottom of the tube and air-dry samples for about 5 min. Ensure that the DNA does not dry out completely.</li>
	<li>Dissolve 80 &#956;g extracted DNA in 21.0 &#956;l of the digestion buffer, add 1 unit of DNase I dissolved in 2.0 &#956;l of digestion buffer.</li>
	<li>Vortex lightly to achieve thorough mixing and incubate for 1.5 hr at 37 &#176;C.</li>
	<li>At the end of the incubation period, add 216.4 &#956;l of dibasic 50 mM Na phosphate solution containing 2.0 mM KCl, 1.0 mM DFO and 200 mM MgCl2.</li>
	<li>Prepare &#8220;digestion buffer-2&#8221; by combining one volume of digestion buffer with nine volumes of dibasic, 50 mM Na phosphate (with 2.0 mM KCl, 1.0 mM DFO and 200 mM MgCl2).</li>
	<li>Add 0.025 units of phosphodiesterase (PDE) I enzyme in 16.3 &#956;l of digestion buffer-2. Pipette up and down for several seconds, then vortex lightly to achieve thorough mixing and incubate for 1.5 hr at 37 &#176;C.</li>
	<li>Add 0.4 units of alkaline phosphatase (AP) in 8.0 &#956;l of digestion buffer-2. Pipette up and down for several seconds, then vortex lightly to achieve thorough mixing and incubate for 1.5 hr at 37 &#176;C. Add 33.0 &#956;l HPLC grade MeOH.</li>
	<li>Run digested DNA samples on HPLC, as described below, right away or store at -80 &#176;C until use to minimize oxidation. &#160;NOTE: The 1.5-hr digestion steps suggested here are based on such intervals suggested elsewhere9 and on the findings that similar protocols achieve complete DNA digestion9. Therefore, it was assumed that the only limiting factor to achieve complete digestion was time and that enzyme inhibition and sample inhomogeneity were negligible.</li>
</ol>
4. HPLC Run: Preparation of Mobile Phase, Instrumentation Setup and Maintenance
<ol>
	<li>Use ultrapure water to prepare all buffer solutions and treated with high bond strength resin (e.g., Chelex 100: styrene divinylbenzene copolymer with iminodiacetate ions that chelate transition metals with high affinity) to minimize metal contamination and DNA oxidation during sample preparation.</li>
	<li>Prepare 250 mM Na phosphate buffer, pH 6.2. Use a ratio of 1 : 3.6957 of dibasic sodium phosphate (FW = 177.99 g/mol) to monobasic sodium phosphate (FW = 119.98 g/mol). 
	Example: Therefore, for a 3 L stock solution combine 70.81 g of monobasic and 28.43 g of dibasic powder, add water up to e.g., 2.8 L and verify that the pH of the solution is 6.2. Adjust pH with 250 mM mono or dibasic sodium phosphate solutions, prepared separately and not by concentrated acids or bases. Add 2.24 g of KCl to obtain a concentration of 10 mM KCl.</li>
	<li>Prepare mobile phase in three bottles, at least 1 L each, containing: solvent B &#8211; HPLC-grade methanol, solvent &#8211; B ultrapure water, and solvent C &#8211; phosphate buffer from step 4.2. Insert HPLC mobile phase supply tubing into the bottles; remaining tubes must be also dipped in one of the bottles (e.g., solution C) and primed even if they are not required for mixing.</li>
	<li>During the run, mix the mobile phase solutions as 6% solvent A, 74% solvent B and 20% solvent C to achieve final solution of 50 mM sodium phosphate buffer (pH 6.2) with 2.0 mM KCl, and 6% MeOH.</li>
	<li>Detach the electrode from the electrochemical detector, disassemble it and clean reference and working electrodes with the electrode cleaning solutions and polishing discs recommended by the manufacturer. Rinse the cleaned surfaces with ultrapure water, wipe the water off gently and ensure the detector is dry before turning the system on.</li>
	<li>Reassemble the detector and connect the mobile phase inlet to the electrode. Turn on the flow and allow the mobile phase to fill up the electrode before attaching the outlet mobile phase tube. Install a new column prior to starting optimization. Ensure that installation is in the proper direction and follows all manufacturer&#8217;s recommendations.</li>
	<li>Turn on the HLPC instrumentation well ahead (e.g., 1 hr) of sample analyses to allow equilibration and to reduce baseline noise. See Table 1 for suggested settings; the back pressure limit should be set to 3,000 psi to avoid column damage. Turn on the degasser. Proceed with priming the pumps and setting up solvent gradient.</li>
	<li>Dry prime the pumps according to manufacturer&#8217;s instructions. Do this to wet the lines when a mobile phase has been replaced or the tubing inlet is exposed to air.</li>
	<li>Locate the priming disc and insert a 10-15 ml plastic syringe there. Make sure the syringe is fully inserted before opening the line. To open the lines simply turn the disc counter clockwise for one turn.</li>
	<li>Start the prime with the appropriate HPLC interface selection. Gently pull the syringe to draw fluid into the lines. Once fluid is flowing, the prime is complete; allow the program to finish the run. Repeat for all lines (usually four, depending on the instrument).</li>
	<li>Alternatively, wet prime when the lines are already wetted from an earlier dry prime (see below). &#160; 
	NOTE: When a large amount of time has elapsed between uses (e.g., 2 weeks or more), a dry prime is recommended.</li>
	<li>Change the composition of the mobile phase solvent components to 25% each using the HPLC user interface. From the interface, select &#8220;Direct Function&#8221; option, then select &#8220;Wet Prime&#8221; option. This will prime all the lines at the same time.</li>
	<li>Make sure all the air is out of the system by observing the wet prime line as it enters the waste container. After a good prime, ensure that no bubbles are left in the line. If bubbles persist, repeat priming.</li>
	<li>Once the pump is primed, begin moving the HPLC mobile phase. Use the HPLC interface to manually change the composition of the mobile phase to 6% methanol (solvent A) and 94% water (solvent B), and select a flow rate of 0.9 ml/min. Allow 5 min to clear any methanol from the column cleaning step.</li>
	<li>After 5 min, change the composition to 6% methanol (solvent A), 74% water (solvent B), 20% buffer (solvent C), increase the flow rate to 1.0 ml/min. Set other parameters as summarized in Table 1, and set the back pressure limit to 3,000 psi to avoid column damage. &#160; 
	NOTE: Potential problems with HPLC setup include drifting baseline, split peak and diminished signal intensity. These can usually be overcome by cleaning the column after each use, frequent electrode cleaning, and ensuring that the buffer solutions are free of contamination (e.g., particulate matter or microbial growth).</li>
	<li>Run the mobile phase for about 1-1.5 hr to achieve a stable baseline signal.</li>
	<li>Using the instrument software interface, select parameters as indicated in Table 1 and set run time to 15 min. Inject the sample. Use increased run times for complex samples (determine this empirically by observing the presence or absence of peaks beyond 15-min interval). &#160; 
	NOTE: Instrument software allows programming of sample run conditions and setup for autosampling.</li>
	<li>After the runs have been completed, turn off the detector cell using the interface of the detector. It is important to follow manufacturer&#8217;s recommendations regarding powering down the detector as improper handling and shut-down can damage the instrument. DO NOT turn off the cell by switching the back of the detector as this could damage the instrument.</li>
	<li>Manually change the mobile phase composition to 6% methanol and 94% water. Run for 5 min.</li>
	<li>Manually reduce the flow rate to 0.7 ml/min, immediately change the composition to 50% MeOH and 50% water. Run for at least 20 min. Failure to run this step for the recommended time could result in damage to the column and the detector. Turn off the flow, and then turn off the degasser.</li>
</ol>
5. Preparation of Standards
<ol>
	<li>Prepare mobile phase buffer as outlined in steps 4.1&#8211;4.3 above. It is important to use the HPLC mobile phase for preparation of standards to avoid peaks resulting from mixing of dissimilar solutions after sample injection.</li>
	<li>Dilute this solution one in five with ultrapure water prior to use, and the final solution must contain 6% MeOH.</li>
	<li>dGuo Standard 
	NOTE: High oxidation potential required for dGuo detection may increase the risk of measuring interfering electroactive compounds. This can be verified by, for example, a standard curve of UV detection of dGuo at 260 nm and comparing it to the standard curve created with EC detection. If both align well (e.g., Supplementary Figure 1) and the sample matrix effects have been taken into account, one may proceed with dGuo detection by UV at 260 nm, rather than EC detection.
	<ol>
		<li>Measure 1.0 mg of dGuo in a 1.5 ml microcentrifuge tube. Add 1.0 ml HPLC grade MeOH.</li>
		<li>Vortex at high speed until dGuo dissolves completely. This is the primary stock; store at -20 &#176;C for several weeks.</li>
		<li>To create the secondary dGuo stock (0.5 mM), dilute 143 &#181;l of primary dGuo stock in 857 &#181;l of HPLC mobile phase. Store on ice until use and prepare fresh daily.</li>
		<li>Make further dilutions to create a standard curve (e.g., Table 2). Store solutions on ice and prepare fresh daily. Run standards in series with experimental samples.</li>
		<li>Prior to run, vary the detector voltage using the highest concentration of the stock to find optimal voltage.</li>
	</ol>
	</li>
	<li>8-oxo-dGuo Standard
	<ol>
		<li>Measure 1.0 mg of 8-oxo-dGuo in a 1.5 ml microcentrifuge tube. Add 0.1 ml HPLC grade MeOH. Vortex on high speed until 8-oxo-dGuo dissolves completely. This is the primary stock of 8-oxo-dGuo. Store at -20 &#176;C for several weeks.</li>
		<li>In a separate tube, combine 2.9 &#956;l of primary stock and 997.1 &#956;l of HPLC mobile phase buffer, vortex. This is the secondary stock (10,000 nM).</li>
		<li>To create a working solution for the standard curve (250 nM), combine 24.4 &#956;l of secondary stock and 975.6 &#956;l of mobile phase buffer.</li>
		<li>Perform further dilutions to provide the solutions required to create a standard curve (e.g., Table 3). Store solutions on ice and prepare fresh daily. Run standards with the experimental samples.</li>
	</ol>
	</li>
	<li>Quantification
	<ol>
		<li>Integrate the area under the curve for both 8-oxo-dGuo and dGuo using standard software (as a part of HPLC-computer interface; see Supplementary Figure 2A).</li>
		<li>Construct standard curve (known concentration of each analyte vs. area under the curve (Supplementary Figure 2B). Using the equation of the standard curve, calculate the ratio for 8-oxo-dGuo/dGuo for each sample.</li>
	</ol>
	</li>
</ol>
6. Agarose Gel Electrophoresis
NOTE: Agarose gel electrophoresis may be performed to verify the completeness of DNA digestion.
<ol>
	<li>Prepare 50x Tris-acetate-EDTA (TAE) buffer, by dissolving 242 g Tris base (FW = 121.14) in 750 ml ultrapure water. Add 57.1 ml glacial acetic acid and 100 ml of 0.5 M EDTA (pH 8.0; EDTA will completely dissolve when the solution pH is adjusted to 8.0 with e.g., NaOH) and add ultrapure water to a final volume of 1 L. Store at RT, dilute this solution 50x prior to use.</li>
	<li>Weigh out 1 g of agarose and dissolve it in 100 ml 1x TAE buffer, heating in the microwave for 1-2 min. Wear goggles and protective equipment to avoid contact with boiling agarose.</li>
	<li>Cool the solution down until approximately 50 &#176;C, add 5 &#956;l ethidium bromide (caution: mutagen) and pour it into the agarose gel running apparatus. Insert the comb.</li>
	<li>Wait until the solution solidifies, pour TAE buffer over the gel and load the samples (volume depends on the comb size; typically, 10-20 &#956;l of DNA sample is loaded, mixed with 6x running dye to visualize and facilitate loading).</li>
	<li>Run the gel until the dye migrates approximately 2/3 the length of the gel (e.g., 45 min at 150 V). Visualize DNA bands under the UV light.</li>
</ol>

<ol><li>Helbock, H. J., Beckman, K. B., Shigenaga, M. K., Walter, P. B., Woodall, A. A., Yeo, H. C., Ames, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+oxidation+matters%3A+The+HPLC-electrochemical+assay+of+8-oxo-deoxyguianosine+and+8-oxo-guanine%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci%22%5BJournal%5D)">DNA oxidation matters: The HPLC-electrochemical assay of 8-oxo-deoxyguianosine and 8-oxo-guanine.</a> Proc. Natl. Acad. Sci. 95 (1), 288-293 (1998).</li>
<li>Valavanidis, A., Vlachogianni, T., Fiotakis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((8-hydroxy-2%27+-deoxyguanosine+%288-OHdG%29%3A+A+critical+biomarker+of+oxidative+stress+and+carcinogenesis%5BTitle%5D)+AND+%22J.+Environ.+Sci+Health+C+Environ.+Carcinog.+Ecotoxicol.+Rev%22%5BJournal%5D)">8-hydroxy-2' -deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis.</a> J. Environ. Sci Health C Environ. Carcinog. Ecotoxicol. Rev. 27 (2), 120-139 (2009).</li>
<li>Cadet, J., Bellon, S., Douki, T., Frelon, S., Gasparutto, D., Muller, E., Pouget, J. P., Ravanat, J. L., Romieu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radiation-induced+DNA+damage%3A+formation%2C+measurement%2C+and+biochemical+features%5BTitle%5D)+AND+%22J+Environ+Pathol+Toxicol+Oncol%22%5BJournal%5D)">Radiation-induced DNA damage: formation, measurement, and biochemical features.</a> J Environ Pathol Toxicol Oncol. 23 (1), 23-23 (2004).</li>
<li>Weiss, J. M., Goode, E. L., Ladiges, W. C., Ulrich, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polymorphic+variation+in+hOGG1+and+risk+of+cancer%3A+a+review+of+the+functional+and+epidemiologic+literature%5BTitle%5D)+AND+%22Mol.+Carcinog%22%5BJournal%5D)">Polymorphic variation in hOGG1 and risk of cancer: a review of the functional and epidemiologic literature.</a> Mol. Carcinog. 42 (3), 127-141 (2005).</li>
<li>Culp, S. J., Cho, B. P., Kadlubar, F. F., Evans, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+Conformational+Analyses+of+8-hydroxy-2%27-deoxyguanosine%5BTitle%5D)+AND+%22Chem.+Res.+Toxicol%22%5BJournal%5D)">Structural and Conformational Analyses of 8-hydroxy-2'-deoxyguanosine.</a> Chem. Res. Toxicol. 2 (6), 416-422 (1989).</li>
<li>Cooke, M. S., Loft, S., Olinski, R., Evans, M. D., Bialkowski, K., Wagner, J. R., Dedon, P. C., Møller, P., Greenberg, M. M., Cadet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recommendations+for+standardized+description+of+and+nomenclature+concerning+oxidatively+damaged+nucleobases+in+DNA%5BTitle%5D)+AND+%22Chem.+Res.+Toxicol%22%5BJournal%5D)">Recommendations for standardized description of and nomenclature concerning oxidatively damaged nucleobases in DNA.</a> Chem. Res. Toxicol. 23 (4), 705-707 (2010).</li>
<li>Jang, Y. H., Goddard, W. A. 3rd, Noyes, K. T., Sowers, L. C., Hwang, S., Chung, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+principles+calculations+of+the+tautomers+and+pKa+values+of+8-oxoguanine%3A+implications+for+mutagenicity+and+repair%5BTitle%5D)+AND+%22Chem.+Res.+Toxicol%22%5BJournal%5D)">First principles calculations of the tautomers and pKa values of 8-oxoguanine: implications for mutagenicity and repair.</a> Chem. Res. Toxicol. 15 (8), 1023-1035 (2002).</li>
<li>Park, J. -H., Gopishetty, S., Szewczuk, L. M., Troxel, A. B., Harvey, R. G., Penning, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+of+8-oxo-7%2C8-dihydro-2%27-deoxyguanosine+%288-oxo-dGuo%29+by+PAH+o-quinones%3A+involvement+of+reactive+oxygen+species+and+copper%28ii%29%2Fcopper%28i%29+redox+cycling%5BTitle%5D)+AND+%22Chem.+Res.+Toxicol%22%5BJournal%5D)">Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) by PAH o-quinones: involvement of reactive oxygen species and copper(ii)/copper(i) redox cycling.</a> Chem. Res. Toxicol. 18 (6), 1026-1037 (2005).</li>
<li>Mangal, D., Vudathala, D., Park, J. H., Lee, S. H., Penning, T. M., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+7%2C8-dihydro-8-oxo-2%27-deoxyguanosine+in+cellular+DNA+during+oxidative+stress%5BTitle%5D)+AND+%22Chem.+Res.+Toxicol%22%5BJournal%5D)">Analysis of 7,8-dihydro-8-oxo-2'-deoxyguanosine in cellular DNA during oxidative stress.</a> Chem. Res. Toxicol. 22 (5), 788-797 (2009).</li>
<li>Ravanat, J. L., Douki, T., Duez, P., Gremaud, E., Herbert, K., Hofer, T., Lasserre, L., Saint-Pierre, C., Favier, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+background+level+of+8-oxo-7%2C8-dihydro-2%27-deoxyguanosine%3A+an+isotope+based+method+to+evaluate+artefactual+oxidation+of+DNA+during+its+extraction+and+subsequent+work-up%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">Cellular background level of 8-oxo-7,8-dihydro-2'-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up.</a> Carcinogenesis. 23 (11), 1911-1918 (2002).</li>
<li>Gossen, J. A., De Leeuw, W. J. F., Tan, C. H. T., Zwarthoff, E. C., Berends, F., Lohman, P. H. M., Knook, D. L., Vijg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+rescue+of+integrated+shuttle+vectors+from+transgenic+mice%3A+A+model+for+studying+mutations+in+vivo%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Efficient rescue of integrated shuttle vectors from transgenic mice: A model for studying mutations in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 86 (20), 7971-7975 (1989).</li>
<li>Test No. 488: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays. , Organisation for Economic Co-operation and Development (OECD). Available from: 
 <a target="_blank" href="http://www.oecd-ilibrary.org">http://www.oecd-ilibrary.org</a> (2015).</li>
<li>Van Campen, L. E., Murphy, W. J., Franks, J. R., Mathias, P. I., Toraason, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidative+DNA+damage+is+associated+with+intense+noise+exposure+in+the+rat%5BTitle%5D)+AND+%22Hear+Res%22%5BJournal%5D)">Oxidative DNA damage is associated with intense noise exposure in the rat.</a> Hear Res. 164 (1-2), 164-161 (2002).</li>
<li>European Standards Committee on Oxidative DNA Damage (ESCODD). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+DNA+oxidation+in+human+cells+by+chromatographic+and+enzymic+methods%5BTitle%5D)+AND+%22Free+Radic.+Biol.+Med%22%5BJournal%5D)">Measurement of DNA oxidation in human cells by chromatographic and enzymic methods.</a> Free Radic. Biol. Med. 34 (8), 1089-1099 (2003).</li>
<li>Rebelo, I. A., Piedade, J. A., Oliveira-Brett, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+an+HPLC+method+with+electrochemical+detection+of+femtomoles+of+8-oxo-7%2C8-dihydroguanine+and+8-oxo-7%2C8-dihydro-2%27-deoxyguanosine+in+the+presence+of+uric+acid%5BTitle%5D)+AND+%22Talanta%22%5BJournal%5D)">Development of an HPLC method with electrochemical detection of femtomoles of 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of uric acid.</a> Talanta. 63 (2), 323-331 (2004).</li>
<li>Ravanat, J. -L., Turesky, R. J., Gremaud, E., Trudel, L. J., Stadler, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+8-oxoguanine+in+DNA+by+gas+chromatography-mass+spectrometry+and+HPLC-electrochemical+detection%3A+overestimation+of+the+background+level+of+the+oxidized+base+by+the+gas+chromatography-mass+spectrometry+assay%5BTitle%5D)+AND+%22Chem.+Res.+Toxicol%22%5BJournal%5D)">Determination of 8-oxoguanine in DNA by gas chromatography-mass spectrometry and HPLC-electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography-mass spectrometry assay.</a> Chem. Res. Toxicol. 8 (8), 1039-1045 (1995).</li>
<li>Kawanishi, S., Murata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+DNA+damage+induced+by+bromate+differs+from+general+types+of+oxidative+stress%5BTitle%5D)+AND+%22Toxicology%22%5BJournal%5D)">Mechanism of DNA damage induced by bromate differs from general types of oxidative stress.</a> Toxicology. 221 (2-3), 172-178 (2006).</li>
<li>Tahara, S., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Susceptibility+of+mouse+splenic+cells+to+oxidative+DNA+damage+by+x-ray+irradiation%5BTitle%5D)+AND+%22Biol.+Pharm.+Bull%22%5BJournal%5D)">Susceptibility of mouse splenic cells to oxidative DNA damage by x-ray irradiation.</a> Biol. Pharm. Bull. 27 (1), 105-108 (2004).</li>
<li>Garratt, L. W., Mistry, V., Singh, R., Sandhu, J. K., Sheil, B., Cooke, M. S., Sly, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interpretation+of+urinary+8-oxo-7%2C8-dihydro-2%27-deoxyguanosine+is+adversely+affected+by+methodological+inaccuracies+when+using+a+commercial+ELISA%5BTitle%5D)+AND+%22Free+Radic.+Biol.+Med%22%5BJournal%5D)">Interpretation of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine is adversely affected by methodological inaccuracies when using a commercial ELISA.</a> Free Radic. Biol. Med. 48 (11), 1460-1464 (2012).</li>
<li>Cooke, M. S., Collins, A., Olinski, R., Rozalski, R., Loft, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Harmonising+measurements+of+8-oxo-7%2C8-dihydro-2%E2%80%B2-deoxyguanosine+in+cellular+DNA+and+urine%5BTitle%5D)+AND+%22Free+Radic.+Res%22%5BJournal%5D)">Harmonising measurements of 8-oxo-7,8-dihydro-2′-deoxyguanosine in cellular DNA and urine.</a> Free Radic. Res. 46 (4), 541-553 (2012).</li>
<li>Cadet, J., Douki, T., Ravanat, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+oxidatively+generated+base+damage+in+cellular+DNA%5BTitle%5D)+AND+%22Mutat+Res%22%5BJournal%5D)">Measurement of oxidatively generated base damage in cellular DNA.</a> Mutat Res. 711 (1-2), 3-12 (2011).</li>
<li>Chomczynski, P., Mackey, K., Drews, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNAzol%3A+a+reagent+for+the+rapid+isolation+of+genomic+DNA%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">DNAzol: a reagent for the rapid isolation of genomic DNA.</a> Biotechniques. 22 (3), 550-553 (1997).</li>
<li>Collins, A. R., Cadet, J., Möller, L., Poulsen, H. E., Viña, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Are+we+sure+we+know+how+to+measure+8-oxo-7%2C8-dihydroguanine+in+DNA+from+human+cells%5BTitle%5D)+AND+%22Arch+Biochem+Biophys%22%5BJournal%5D)">Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells.</a> Arch Biochem Biophys. 423 (1), 57-65 (2004).</li>
<li>Badouard, C., Ménézo, Y., Panteix, G., Ravanat, J. L., Douki, T., Cadet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+new+types+of+DNA+lesions+in+human+sperm%5BTitle%5D)+AND+%22Zygote%22%5BJournal%5D)">Determination of new types of DNA lesions in human sperm.</a> Zygote. 16 (1), 9-13 (2008).</li>
<li>Cadet, J., Douki, T., Gasparutto, D., Ravanat, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidative+damage+to+DNA%3A+formation%2C+measurement+and+biochemical+features%5BTitle%5D)+AND+%22Mutat+Res%22%5BJournal%5D)">Oxidative damage to DNA: formation, measurement and biochemical features.</a> Mutat Res. 531 (1-2), 1-2 (2003).</li>
<li>Validation of analytical procedures: text and methodology Q2(R1). International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), 2015 Mar 1, San Diego, CA, , Available at: <a target="_blank" href="http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf">http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf</a> (2015).</li>
</ol>

No conflict of interest or competing financial interests declared.

Although 8-oxo-dGuo has been reported as a useful biomarker of DNA oxidation, its reliable quantification can pose a challenge. Although several published methods exist, there is a need for a comprehensive, descriptive overview of protocol to permit researchers to deploy the method in their laboratories. Here we present a detailed overview of an HPLC-based protocol that will permit new users to establish an effective method for 8-oxo-dGuo detection and quantification.
Three major methods that ...

Reactive oxygen species (ROS), whose steady-state levels can increase during many pathological conditions and xenotoxic metabolism, contribute to an increased frequency of oxidative DNA damage. Among several possible nucleobases oxidation products, oxidative DNA damage can readily be measured using the stable marker 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo), which is one of the oxidized forms of 2&#8217;-deoxyguanosine (dGuo)1. 8-oxo-dGuo is the most abundant DNA lesion2 and, therefore, has been studied to greater detail as a DNA oxidation biomarker despite the existence of multiple DNA oxidation products 3. In humans, this damage can be repaired via base excision repair by 8-oxoguanine glycosylase 1 (hOGG1)4. If left unrepaired, 8-oxo-dGuo can contribute to the formation of base pair-substitution mutations (i.e., G to T transversions)4. Importantly, 8-oxo-dGuo is an established marker for DNA damage in relation to the initiation and promotion of carcinogenesis2. Therefore, accurate quantification of 8-oxo-dGuo is a useful and desirable biomarker of oxidative DNA damage5.
There is widespread confusion in the literature regarding the correct names for oxidatively-damaged forms of 2-deoxyguanosine and, moreover, the correct name of the compound(s) routinely measured as a biomarker of oxidative DNA damage6. The 6,8-diketo and 6-enol,8-keto tautomeric forms of 8-oxo-dGuo (shown in Figure 1) are the two most prominent tautomers discussed in the literature5,7. The 6,8-diketo form is the most prominent form at physiological pH of 7.4, and is the most prominent DNA oxidation product7. Therefore, 8-oxo-dGuo, rather than 8-hydroxy-dGuo is the most appropriate name for this oxidation product6. It is also important to note that 2-deoxyguanosine (dGuo), rather than nucleobase guanine (Gua) or ribonucleoside guanosine (Guo), respectively, is detected by most methods6.
Accurate detection and quantification of 8-oxo-dGuo is challenging due to: i) variability in the digestion of the DNA sample, ii) adventitious oxidation of dGuo to 8-oxo-dGuo that can occur during sample preparation, and iii) the need for effective validation of the analytical HPLC-ED method8. In this protocol, we aimed at achieving i) by providing conditions, favorable for complete DNA digestion and ii) by the inclusion metal chelator and chelator-treated solutions and a special DNA-isolating reagent, while iii) was only partially addressed by inclusion of positive controls and thus providing that the method is capable of detecting 8-oxo-dGuo in biological samples. Further validation is beyond the scope of this paper. However, we are confident that this protocol will help the prospective users determine the extent to which they need to formally validate the protocol, depending on their purposes. A list of steps required for the formal validation of the method is provided further. During the development and deployment of a method for 8-oxo-dGuo detection, published methods were reviewed and consolidated. Thus, this method eliminates the need to gather information from several published sources that often lack important experimental details while also providing rapid and straightforward means of testing if the method for the detection and quantification of 8-oxo-dGuo has been adopted successfully. This adapted method was employed to successfully analyze DNA samples from cultured cells and murine tissue. This video article will assist other groups in establishing an effective method for reliable detection and quantification of 8-oxo-dGuo by HPLC-ED.

<table><tbody><tr><td>8-oxo-dGuo standard</td><td>Cayman Chemical Company</td><td>89320</td><td>Inappropriately referred to as &quot;8-hydroxy-2&#039;-deoxy Guanosine&quot;; see &lt;strong&gt;Fig. 1&lt;/strong&gt; and text for details.</td></tr><tr><td>Alkaline phosphatase</td><td>Sigma-Aldrich</td><td>P5931</td><td>From &lt;em&gt;E. coli&lt;/em&gt;</td></tr><tr><td>Chelex 100</td><td>Sigma-Aldrich</td><td>C7901</td><td>Chelates heavy metals</td></tr><tr><td>Desferoxamine mesylate</td><td>Sigma-Aldrich</td><td>D9533</td><td></td></tr><tr><td>dGuo standard</td><td>Sigma-Aldrich</td><td>D7145</td><td></td></tr><tr><td>Dibasic sodium phosphate</td><td>Sigma-Aldrich</td><td>S9390</td><td></td></tr><tr><td>DNA from salmon sperm</td><td>Sigma-Aldrich</td><td>D1626</td><td>Sodium salt</td></tr><tr><td>DNase I</td><td>Sigma-Aldrich</td><td>D4527</td><td>TypeII, from bovine pancreas</td></tr><tr><td>DNAzol</td><td>Invitrogen</td><td>10503-27</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td><td>Sigma-Aldrich</td><td>E4884</td><td>The compound would not completely dissolve until solution pH is adjusted to 8.0 (&lt;em&gt;e.g.&lt;/em&gt; with NaOH)</td></tr><tr><td>F12-K media</td><td>ATCC</td><td>30-2004</td><td></td></tr><tr><td>Foetal bovine serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Guard column</td><td>Chromatographic Specialties</td><td>YBA 99S03 0204GC</td><td>Protects colum from contamination; may also lead to pressure build-up</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Monobasic sodium phosphate</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Invitrogen</td><td>15190-250</td><td></td></tr><tr><td>Phosphodiesterase I enzyme&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P3243</td><td>Type II from Crotalus adamaneus venom</td></tr><tr><td>Teflon homogenizer</td><td>Thomas Scientific</td><td>7724T-1 or 7724T-5 for 1 or 5 mL, respectively</td><td>Volume (holding capacity) depends on the amount of sample to be processed.</td></tr><tr><td>Trypsin</td><td>Invitrogen</td><td>15050-065</td><td></td></tr><tr><td>YMC-BASIC column with bonded spherical silica</td><td>Chromatographic Specialties</td><td>YBA 99S03 1546WT</td><td></td></tr></tbody></table>

hplc measurement of the dna oxidation biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in cultured cells and animal tissues

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2&#39;-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxo-dGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO3, and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples.

dGuo was observed to have a retention time of 4.7 min whereas 8-oxo-dGuo had a retention time of approximately 6.4 min (Figure 2A and B). There is about 1,000-fold difference in the peak heights between the two analytes, as seen in Figure 2C. Voltammograms for 8-oxo-dGuo and dGuo were obtained by running standards at a working potential in the range of +0.2 to +1.1 V. The optimum working potential for 8-oxo-dGuo was determined to be +0.5 V, and +0.9 V for dGuo (F...

The goal of this protocol is the detection of the DNA oxidation marker, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo) by HPLC-ED, in DNA from cultured cells or animal tissues.

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

The overall goal of this procedure is to detect and quantify the DNA oxidation product eight OXO DG in biological samples by high pressure liquid chromatography with electrochemical detection or H-P-L-C-E-D. This is accomplished by isolation of a DNA sample from biological samples, followed by complete DNA digestion in a manner that minimizes undesirable DNA oxidation that can occur during sample preparation through the inclusion of metal chelator and a special DNA isolating reagent. Next H-P-L-C-E-D run conditions are optimized and standard curves for eight OXO DG and two primed dioxazine are constructed.The final step is to run digestive DNA in series with pure eight OD DG and two prime deoxy guine in the HPLC in order to find the eight OD DG to two prime deoxy guine ratios in the digested DNA samples under investigation. Ultimately eight OD DG detection and quantification by H-P-L-C-E-D is used to quantify the extent of DNA oxidation in biological samples. Yeah, the main advantage of this technique over existing methods is that it consolidates all relevant information from several published sources in addition to providing rapid and straightforward means of testing the success of the deployment of this method in a new laboratory setting.This method can help answer key questions in biomedical and toxicology research, such as how oxidative stress, or more specifically DNA oxidation is mechanistically linked to adverse effects and cultured cells are experimental animals. Generally, people new to this technique will struggle because there are many parameters that need to be optimized in a new laboratory setting in order to ensure that the new method is working correctly. Visual demonstration of this method is critical as there are experimental steps can be difficult to learn since there are numerous parameters that need to be optimized.Dr.Nikolai, she, Dean Kennedy, our former co-op student from the University of Ottawa, will be demonstrating this procedure. DNA can be extracted from cultured cells. To do this, verify that there is enough cells for DNA extraction, usually at least two to three 10 centimeter plates with a cell density of about 70%Discard the cell culture media and wash the cells with PBS, scrape or trypsin the cells off the plate and put them in a 50 milliliter conical polystyrene centrifuge tube harvest by centrifugation cells form a pellet at the bottom of the tube.Homogenized cells in a 50 milliliter conical polystyrene centrifuge tube with one milliliter of DNA isolating agent such as DN Azo. Use a 1000 microliter pipette tip to disperse the cell pellet until the solution is homogenous. Transfer the homogenate to a new 1.5 milliliter micro centrifuge tube and incubate on ice for 10 to 20 minutes.Alternatively, if working from tissue homogenize 15 to 20 milligrams of tissue in a one milliliter handheld non-stick glass homogenizer containing 500 microliters of DNA isolating agent, gently raise and lower the homogenizer for about one minute. Softer tissues will require less time. Gentle treatment ensures less shearing of DNA store the homogenate on ice for about 10 to 20 minutes.Next palate, the homogenate derived from either cells or tissue by centrifugation for 10 minutes at 10, 000 G and four degrees Celsius in a 1.5 milliliter conical micro centrifuge tube. Carefully transfer the resulting supinate to a new 1.5 milliliter micro centrifuge tube. Paying careful attention to avoid contact with the pellet precipitate the DNA from the homogenate by adding 0.5 milliliters of 100%ethanol per one milliliter of homogenate.Invert the tubes 10 times to ensure the isolating reagent and the ethanol are sufficiently mixed. At this point, the DNA precipitate is viscous. Spool it onto a plastic pipette tip and then transfer it to a new 1.5 milliliter conical centrifuge tube spooling may not work for all tissues and centrifugation may be required.Next, add one milliliter of 75%ethanol to the isolated DNA and resus. Suspend the DNA pellet thoroughly by inverting the tubes 10 times. Carefully decant the ethanol from the tube.Ensure that the DNA pellet sticks to the side of the tube. Repeat the DNA wash once more and either store the sample in ethanol at minus 20 degrees Celsius or immediately proceed to digestion. DNA samples can be stored in ethanol for future use at negative 20 degrees Celsius for several months prior to use.Remove ethanol by storing tubes vertically for one to two minutes or removing ethanol by a pipet from the bottom of the tube. Dissolve the DNA in digestion and then quantify using standard spectroscopic methods, remove any remaining ethanol from the bottom of the tube and air dry the samples for about five minutes. Ensure that the DNA does not dry out completely dissolve 80 micrograms of the extracted DNA in 21 microliters of the digestion buffer.Then add one unit of DNA one dissolved in two microliters of digestion. Buffer to the sample vortex lightly to achieve thorough mixing before incubating for 1.5 hours at 37 degrees Celsius. At the end of the incubation period, add 216.4 microliters of digestion buffer two.Next, add 0.0 25 units of phosphodiesterase, one enzyme in 16.3 microliters of digestion. Buffer two to the sample pipette up and down for several seconds after vortexing lightly to achieve thorough mixing. Incubate the sample for 1.5 hours at 37 degrees Celsius.Following the incubation add 0.4 units of alkaline phosphatase in eight microliters of digestion. Buffer two to the sample pipette up and down for several seconds and vortex lightly to achieve thorough mixing. After incubating for 1.5 hours at 37 degrees Celsius, add 33 microliters of HPLC grade methanol to the sample.Prepare mobile phase buffer as described in the text protocol. It is important to use the HPLC mobile phase for preparation of the standards to avoid peaks resulting from mixing of dissimilar solutions After sample injection, dilute the solution one in five with ultrapure water prior to use and the final solution must contain 6%methanol. To prepare the two prime deoxy Guine standard measure one milligram of two prime deoxy guine in a 1.5 milliliter micro centrifuge tube.After adding one milliliter of HPLC grade methanol vortex at high speed until two prime deoxy guine dissolves completely to create the secondary two prime deoxy Guine stock dilute 143 microliters of primary two prime deoxy guine stock in 857. Microliters of HPLC mobile phase. Make further dilu to create a standard curve store the solutions on ICE and prepare fresh daily.Prior to the HPLC run, vary the detector voltage using the highest concentration of the stock to find the optimal voltage. Using the HPLC software interface, select parameters as listed in the text protocol and set the runtime to 15 minutes. Inject the sample or standard and run the standards in series with experimental samples.Next, prepare the eight OXO DG standard by adding one milligram of eight OXO DG in a 1.5 milliliter micro centrifuge tube. Alternatively, a commercially available solution can be used after adding one milliliter of HPLC grade methanol vortex on high speed until the eight OXO DG dissolves completely. This is the primary stock of eight OXO DG in a separate tube combined 2.9 microliters of primary stock and 997.1 microliters of HPLC mobile phase buffer and vortex.This is the secondary stock. Then create a working solution for the standard curve by combining 24.4 microliters of the secondary stock and 975.6 microliters of the mobile phase buffer. Perform further dilutions to provide the solutions required to create a standard curve.Run the standards with the experimental samples on the HPLC as before. After running the samples manually reduce the flow rate to 0.7 milliliters per minute and immediately change the composition to 50%methanol and 50%water run for at least 20 minutes. Failure to run this step for the recommended time could result in damage to the column and the detector.Turn off the flow and then turn off the desser. Integrate the area under the curve for both eight Oxo DG and two prime dioxazine using standard software and construct the standard curve. Using the equation of the standard curve, calculate the ratio for eight OXO DG two prime dioxazine for each sample shown.Here are the structures of the two prime DIOXAZINE oxidation product eight OXO dg, and it's TTR eight hydroxyl two prime Dioxazine eight OXO DG is predominant at physiological pH and yet it's TTR eight. Hydroxyl two. Prime dioxazine is often erroneously referred to as the major oxidation product of two prime deoxy guine in publications and vendor catalogs.The retention time for two prime dioxazine is 4.7 minutes, and the retention time for eight OD DG is 6.4 minutes. The two prime dioxazine peak is of much larger intensity than that of eight OXO dg as shown here. The detector voltage can be optimized by varying voltage and plotting the peak area as a function of voltage.Next, a standard curve for both two prime dioxazine and eight Oxo DG should be constructed by varying their concentrations. This should be done in parallel with sample runs. Further buffer composition and DNA digestion time can be optimized by agros gel electrophoresis and HPLC.The assumptions here are that the production of fragments that are too small to be detected on the gel enzyme inhibition and sample in homogeneity are negligible and digestion time is the only limiting factor as shown here. Chromatograms from biological samples are more complex that those from standard curves. However, two prime dioxazine and eight OXO DG peaks can be discerned by their retention time.Once mastered, this technique can be done within a few hours if it is performed properly. Oh, while attempting this procedure, it's important to remember to take all necessary precautions to prevent artifactual DNA oxidation prior to the HPL serum. Following this procedure, other genetic toxicology assays can be performed to determine if DNA oxidation can lead to mutations After its development.This technique paved the way for researchers to quantify DNA oxidation in multiple biological samples, including cultured cells, animal tissues, and human samples. Don't forget that working with many chemicals that lead to DNA oxidation can be extremely hazardous, and precautions such as basic safety techniques, including protective equipment, should always be taken while performing this procedure.

hplc-measurement-dna-oxidation-biomarker-8-oxo-78-dihydro-2

Research

JoVE Journal

Chemistry

Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.

Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2'-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2'-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

This protocol describes an efficient method for quantitatively detecting DNA oxidative damage in MCF-7 cells by ELISA technology.

Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

A protocol to avoid the oxidation of metallic substrates during sample transfer from an inhibited acidic solution to an X-ray photoelectron spectrometer is presented.

Determining the Chemical Composition of Corrosion Inhibitor/Metal Interfaces with XPS: Minimizing Post Immersion Oxidation

We describe the process of fabrication and testing of photonic thermometers.

Fabrication and Testing of Photonic Thermometers

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Learn to quantify DNA oxidation using HPLC-ED to detect 8-oxo-dGuo in cultured cells and animal tissues. This method minimizes artifactual oxidation during sample preparation for reliable biomarker assessment.

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2’-deoxyguanosine, in Cultured Cells and Animal Tissues

Explore More Videos