Sample preparation
Q: Do I need to extract DNA if using urine, plasma, or serum?
A: It is not necessary to extract DNA if samples are a body fluid such as urine, plasma, serum, cerebrospinal fluid, or saliva. Samples may need to be briefly spun down if there are insoluble particles in the sample, otherwise they can be used directly without any pretreatment. For cell and tissue samples, DNA must be extracted first.
Q: How much DNA do I need to use for each assay?
A: 2 ug of completely digested DNA is required for each assay. Here is why:
- Based on the literature, there is about one 8-OHdG site in 100,000 dG sites
- Molecular Weight of 8-OHdG, dA, dT, dC and dG is about 300.
- Our kit has a detection sensitivity of 100 pg/mL
Calculations:
100 pg/mL x 100,000 x 4 (you have an even amount of dA, dT, dC and dG sites) = 4 x 107 pg/mL = 40 ug/mL
In each assay, you use 50 uL of digested DNA sample, therefore, 50 uL x 40 ug/mL = 2 ug.
Q: Can I use less than 2 µg of DNA per sample required?
A: Based on the frequency of 8-OHdG in genomic DNA, at least 2 µg digested DNA is required per assay. Less than 2 µg can be used for samples that have high 8-OHdG levels, but in general, at least 2 µg DNA should be used.
Q: How do I prepare DNA samples?
A: DNA samples must first be converted from dsDNA to ssDNA by heat denaturation, followed by digestion to single nucleotides using nuclease P1. Finally, single nucleotides are converted to single nucleosides by alkaline phosphatase.
Q: What concentration of nuclease P1 and alkaline phosphatase do you recommend?
A: We recommend resuspending DNA to 1-5 mg/ml, using 5-20 units of nuclease P1, and 5-10 units of alkaline phosphatase. The minimum amount of DNA is 2ug/well, but you can always add more than that.
Q: Which nuclease P1 and alkaline phosphatase do you recommend?
A: We recommend nuclease P1 from Sigma #N8630 and alkaline phosphatase from Sigma #P5931.
Q: Do all samples need to be treated with nuclease and alkaline phosphatase?
A: Plasma, serum or urine samples can be assayed directly without the enzyme treatment. The nuclease and alkaline phosphatase treatments are only needed for extracted DNA.
Q: Is there an alternative enzyme to nuclease P1?
A: We have not tested any enzymes other than Nuclease P1 to digest DNA. You can try using a different enzyme but only if you can guarantee that it will digest DNA down to the single nucleotide level, which may take some optimization. Some alternate nucleases such as S1 will only partially digest DNA. This is critical, because if you have even dinucleotides present in the sample it will affect the results.
Q: Can I use Shrimp Alkaline Phosphatase during the DNA digestion?
A: Yes, you can use shrimp alkaline phosphatase.
Q: Is an ethanol precipitation step required following nuclease P1 treatment?
A: An ethanol precipitation step is not required at this point because the alkaline phosphates step is performed in 100mM Tris, which is sufficient to bring the pH back to neutral.
Q: How can I confirm that my digestion is complete and I have pure nucleoside samples?
A: The recommended amount of enzyme is sufficient to digest DNA into single nucleosides. Complete digestion can be confirmed by running samples on a PAGE gel, but this is not necessary.
Q: Is this kit suitable for use with bacterial cells?
A: Since this kit uses isolated DNA samples, it is suitable for use with DNA extracted from bacteria.
Q: Can you provide or recommend a DNA extraction kit to use with this kit?
A: We don’t sell DNA extraction kits, however any commercially available kit will work.
Q: Can I use DNA with a concentration lower than the recommended 1-5mg/ml?
A: Yes, as long as the 50uL digested DNA sample that will be used in the assay contains at least 2 µg of DNA.
Q: What volume of DNA should I use?
A: The assay requires 50ul for each well, so the DNA concentration should be maintained at 1-5mg/ml in the volume needed for the assay. For example, if running samples in duplicate, 50ul will be needed for each reaction, plus 10% extra for volume correction, so you would want 110ul.
Q: Does RNA need to be removed from the sample?
A: No additional steps to remove remaining RNA are required. Most DNA extraction kits have an RNase step that is good at removing contaminating RNA and should be followed so that the purified DNA is free of RNA. Although the anti-8-OHdG antibody used in this ELISA will recognize both 8OHdG and 8OHG, any remaining RNA is not likely to contribute to the signal because the antibody used in this kit has about 5 fold higher affinity to 8-OHdG than to 8-OHG.
Q: Can I store DNA in the freezer before using the assay?
A: Unlike other oxidative stress markers, 8-OHdG is very stable byproduct of oxidation and extracted DNA or digested DNA samples can be stored at -20ºC or -80ºC for up to one year.
Q: How should I store urine samples?
A: 8-OHdG is a very stable byproduct of DNA damage. For urine sample, you can store samples at -80ºC for at least one year. 8-OHdG is abundant in urine and samples may need to be diluted several fold before use in this assay.
Q: Can this be used on DNA from the species I am studying?
A: The 8-OHdG modification is universal regardless of species and therefore this kit can be used with DNA from any species.
Q: Can I use this kit on EDTA plasma?
A: The 8-OHdG ELISA kit can be used on plasma samples, and EDTA will not interfere with the assay.
Q: Do serum samples need to be filtered prior to use in the assay?
A: For urine, plasma or serum samples, as long as there are no insoluble particles inside, there is no need to filter them before ELISA steps. They can be used either directly in the assay or diluted with the kit assay diluent. 8-OHdG is abundant in urine, plasma and serum samples, which may require 2-5 fold or higher dilutions.
Q: The protocol suggests diluting high content 8-OHdG samples. Is this referring to all samples, or just ones suspected to have high levels?
A: The 8-OHdG standard curve has a broad range, so a 1:5 sample dilution is recommended as a starting point for the first experiment. After obtaining these preliminary results, the optimal dilution can be determined for further experiments, which may require as much as a 10-20 fold dilution.
Q: What are normal serum levels of 8-OHdG? What are the expected high and low values?
A: The serum levels of 8-OHdG will of course depend on each sample, but typically they will fall within our standard curve, which ranges from 0.078ng/ml-20ng/ml. Occasionally samples will contain higher 8-OHdG levels and will need to be diluted. A preliminary experiment with a 1:5 dilution is recommended to help determine the optimal dilution necessary for preparing remaining samples.
Assay Protocol
Q: Can we use a portion of the plate and then use remaining wells at a later time?
A: The 96-well plate provided with the kit is a strip well plate consisting of 12 8-well strips. Any number of strips can be used for an initial experiment and unused strips can be stored at 4ºC for future use. The wells should be coated fresh each time and the antibody dilutions should be made new for each experiment. A freshly prepared standard curve will need to be run with each experiment.
Q: How can you tell when there is enough color change to stop the reaction?
A: A large incubation range is provided because the development time can vary, which is typical for any ELISA. Upon adding the TMB substrate, a blue color will slowly develop, which will then change to yellow after adding the stop solution. If samples are incubated too long in substrate solution they can become saturated. To determine when to add stop solution, focus on the wells with the standard curve where you will want to see a bright blue color develop for the lowest concentration and a very faint color in the highest concentration. When you see a nice gradient of color you should add the stop solution and read the plate immediately. It is important to add the stop solution to all the wells at the same time, using a multichannel pipettor, regardless of the difference in colors between samples.
Data Analysis
Q: Why does this assay generate a reverse curve?
A: The 8-OHdG ELISA is a competitive ELISA, where any 8-OHdG present in the sample/standards competes with the pre-immobilized 8-OHdG conjugate on the plate for antibody binding. The higher the 8-OHdG levels in the sample or standard, the less antibody binds to the plate and hence the lower OD reading.
Q: How do I make the standard curve and calculate my results?
A: The best way to determine concentrations from a standard curve is to use a 4-parameter curve fitting program, but if you don’t have this software it can be done using Excel. When graphing the standard curve, set the x-axis to logarithmic scale and generate a linear or logarithmic trendline. The standard curves generated with ELISAs are not typically linear, but a linear curve can be created by eliminating the upper and lower values of the curve, as long as the sample values fall within the remaining points. The middle part of the standard curve is the most sensitive and is the best part to use for quantifying samples. The equation of the trendline can be used to calculate the concentrations of unknowns by solving for x in y=mx+b, which is (OD value-b)/m for linear lines or ln(x)=(OD value-b)/m for logarithmic lines.
Troubleshooting
Q: My standard curve OD values are low.
A: Overall low ODs with this assay suggest there was a problem with the coating of the 8-OHdG conjugate onto the plate. If the coating was correct, the 0ng/ml standard would give the highest OD. Here are some suggestions:
- The 8OHdG conjugate is not stable once coated, so it is recommended to use freshly coated plates.
- Aliquot and store the 8-OHdG conjugate at -80ºC and avoid multiple freeze/thaw cycles.
- The substrate solution can take anywhere from 2-30 minutes to completely develop. The gradient developed with the standard curve should be used as an indicator of when to stop the reaction.
- We recommend repeating the same standard concentrations following these recommendations and ensure that the conjugate was properly coated.
Q: What is the most critical step in the procedure?
A: The coating with the conjugate is the most critical step. The 8-OHdG conjugate is not stable once coated, so it is recommended to use freshly coated plates and the wells should be used within 24 hours after coating with 8-OHdG conjugate.