基因组的 8-氧-7,8-二氢-2'-脱氧鸟苷的测量

Antonio  Sarno

doi:10.21769/BioProtoc.878

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Genomic 8-oxo-7,8-dihydro-2'-deoxyguanosine Quantification

基因组的 8-氧-7,8-二氢-2'-脱氧鸟苷的测量

AS Antonio Sarno email

发布: 2013年09月05日第3卷第17期 DOI: 10.21769/BioProtoc.878 浏览次数: 8766

评审: Anonymous reviewer(s)

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Abstract

8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) is among the most common reactive oxygen species-induced DNA lesions and can be used as a biomarker for oxidative stress. The lesion has been linked to several biological processes and diseases, including colorectal cancer, Huntington’s disease, estrogen-induced gene expression, and thymine dimer repair (reviewed in Delaney et al., 2012). The following assay is used to quantify 8-oxo-dGuo levels in DNA as described in Sousa et al. (2013).

Materials and Reagents

NH₄HCO₃ (reagent grade ≥ 99% purity)
MgCl₂(reagent grade ≥ 99% purity)
CaCl₂ (reagent grade ≥ 99% purity)
DNase I from bovine pancreas (F. Hoffmann-La Roche, catalog number: 04716728001 )
Nuclease P1 from P. citrinum (Sigma-Aldrich, catalog number: N8630-1VL )
Phosphodiesterase I from C. adamanteus venom (Sigma-Aldrich, catalog number: P3242-1VL )
Alkaline phosphatase from E. coli (Sigma-Aldrich, catalog number: P5931-100UN )
8-hydroxy-2’-deoxyguanosine (8-oxo-dGuo) (Sigma-Aldrich, catalog number: H5653-1MG )
[¹⁵N₅]-8-hydroxy-2’-deoxyguanosine (Cambridge Isotope Laboratories, catalog number: NLM-6715-0 ) (this is the internal standard)
DNeasy^® Blood & Tissue Kit (QIAGEN, catalog number: 69506 )
LC/MS-grade methanol
Hydrolysis buffer (see Recipes)
Solvent A (see Recipes)
Solvent B (see Recipes)

Equipment

Vortexer
Microcentrifuge
Vacuum centrifuge
LC/MS/MS: We used an LC-20AD HPLC system (Shimadzu Corporation) coupled to an API 5000 triple-quadrupole mass spectrometer (Applied Biosystems)
Zorbax SB-C18 reverse phase chromatography column (2.1 x 150 mm, i.d., 3.5 μm) (Agilent Technologies)

Procedure

English

中文翻译

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Sarno, A. (2013). Genomic 8-oxo-7,8-dihydro-2'-deoxyguanosine Quantification. Bio-protocol 3(17): e878. DOI: 10.21769/BioProtoc.878.