8-OHdG assay:
1) Urine or plasma sample are very easy to assay, they can be used straight or after dilute with assay diluent.
2) In Vitro, 8-OHdG is very stable whether it’s on the DNA or the oxidative byproduct in urine, you can store the digested DNA samples at -20 C or -80 C for long time before assay.
3) For DNA sample, based on publications, about one 8-OHdG site in 10^5 dG sites, from that, here is the calculation:
Assumptions:
Based on literatures, about one 8-OHdG site in 100,000 dG sites
Molecular Weight of 8-OHdG, dA, dT, dC and dG is about 300.
STA-320 has a detection sensitivity of 100 pg/mL
Calculations:
100 pg/mL x 100,000 x 4 (you have even amount of dA, dT, dC and dG sites) = 4 x 10^7 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.
Conclusion: you should use minimal of 2 ug completely digested DNA sample for each assay.
Method from STA-320 Manual:
1.
Extract DNA from cell or tissue samples by a desired method or commercial DNA
Extraction kit.
2.
Dissolve extracted DNA in water at 1-5 mg/mL.
3.
Convert DNA sample to single-stranded DNA by incubating the sample at 95
0
C for
5 minutes and rapidly chilling on ice.
4.
Digest DNA sample to nucleosides by incubating the denatured DNA with 5-20 units of nuclease P1 for 2 hrs at 37 0 C in 20 mM Sodium Acetate, pH 5.2, and following with treatment of 5-10 units of alkaline phosphatase for 1 hr at 37
0
C in
100 mM Tris, pH 7.5.
5.
The reaction mixture is centrifuged for 5 minutes at 6000 g and the supernatant is used for the 8-OHdG ELISA assay.
Example:
1.
Extract DNA from cell or tissue samples by a desired method or commercial DNA
Extraction kit. Dissolve extracted DNA in water at 1 mg/mL.
2.
Add 135 µL of DNA sample in a tube and convert DNA sample to single-stranded
DNA by incubating the sample at 95
0
C for 5 minutes and rapidly chilling on ice.

3.
Add 15 µL of 200 mM Sodium Acetate, pH 5.2 and 5 units of nuclease P1 (Sigma
N8630) to the denatured DNA sample and incubate for 2 hrs at 37
0
C. pH 5.2.
4.
Add 15 µL of 1M Tris, pH 7.5, and 5 units of alkaline phosphatase (Sigma P5931), and incubate for 1 hr at 37
0
C.
5.
The reaction mixture is centrifuged for 5 minutes at 6000 g and the supernatant is used for the 8-OHdG ELISA assay or stored at -20 0 C for up to 6 months .
Note: When 50 µL of the nuclease digestion mixture is used in 8-OHdG ELISA (STA-
320), about 40 µg DNA is used.