| Home > Protocols > Detection of 8-OHdG in tissue samples |
Rev.090309 |
| This is an application example to detect 8-OHdG in tissue samples or cultured cells.
Experimental conditions should be settled by each user. |
| 1) Extract DNA from cells |
Dextract DNA from the homogenates using a DNA extraction kit. Please follow the manufacture's manual.
Followings are DNA extraction kits suitable for 8-OHdG detection, and are available from WAKO Chemicals USA Inc. and from
WAKO Chemicals GmbH (Germany).
1) DNA Extractor® WB Kit (Sodium Iodide Method): cat#291-50502
2) DNA Extractor® TIS Kit (Sodium Iodide Method): cat#296-67701
3) 8-OHdG Assay Preparation Reagent Set: cat#292-67801
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| 2) Calculation of DNA concentration and purity |
| a) |
DNA Solution is diluted to 20-50 µg/mL with TE buffer. |
| b) |
Measure absorbance at wavelength 230nm, 260nm, 280nm and 320nm. |
| c) |
Calculate DNA Concentration. (1.0 of Absorbance at 260nm is equal to 50 µg/mL DNA)
DNA concentration (µg/mL) = (Absorbanve at 260nm) * (50 µg/mL) * dilution rate
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| d) |
Check the purity of DNA sample.
The ratio of (Absorbance at 260nm) : (Absorbance at 280nm) is typically between 1.8 to 1.85.
The purity of isolated DNA is assessed by the ratio of (Absorbance at 260nm) : (Absorbance at 230nm),
and should be between 2.2 to 2.25. |
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| 3) Enzymatic digestion of DNA |
| a) |
Dissolve 200 µg of extracted DNA in 135 µL of water. |
| b) |
Add 15 micro L of 200mM sodium acetate and 6 units of nuclease P1 (15 µL, 1 mg/mL) to
the DNA Solution, and then incubated for 30 min to 1hr at 37 °C after Argon substitution. |
| c) |
Add 1M Tris-HCl buffer (15 µL, pH 7.4) and 2 unit of alkaline phosphatase
(7 µL, 200 units/0.7mL), and incubated for 30 min to 1hr at 37 °C after Argon substitution. |
| d) |
To remove enzymes and other macromolecules, the hydrolysate are filtered through Millipore
Microcon YM-10(catalog#42407) at 14000rpm for 10min. |
| e) |
Apply 50 µL of DNA digest to the wells of ELISA kit. It is recommended that analysis
procedure mentioned here shoukd be completed in one day. |
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NOTE:
1) Enzyme-treated samples should be applied in the same day.
2) TE buffer: 1mM EDTA / 10mM Tris-HCl, pH8.0
3) Water used should be treated by Chelex (Bio Rad) to remove trace metal ions.
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| References |
| 1) |
ML Hamilton, Z Guo, CD Fuller, H Van Remmen, WF Ward, SN Austad, DA Troyer, I Thompson, A Richardson:
A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA.
Nucleic Acids Res. Vol.29(10), p2117-2126, 2001 |
| 2) |
S Toyokuni, et. al.:
Quantitative Immunohistochemical determination of 8-hydroxy-2'-deoxyguanosine by a monoclonal antibody N45.1: Its application to ferric
nitrilotriacetate-induced renal carcinogenesis model.
Laboratory Investigation, Vol. 76, No.3, p365-374, 1997 |
| 3) |
M Kakimoto, T Inoguchi, T Sonta, HY Yu, M Imamura, T Etoh, T Hashimoto and H Nawata:
Accumulation of 8-hydroxy-2'-deoxyguanosine and mitochondrial DNA deletion in kidney of diabetic rats.
Diabetes, Vol. 51, p1588-1595, 2002 |
| 4) |
D Nakae, Y Mizumoto, E Kobayashi, O Noguchi and Y Konishi:
Improved genomic / nuclear DNA extraction for 8-hydroxydeoxyguanosine analysis of small amounts of rat liver tissue.
Cancer Lett., Vol. 97, p233-239, 1995 |
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