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

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.