欢迎您, 登录 | 注册

首页 | English

X
加载中

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).

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

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

癌症生物学 > 通用技术 > 生物化学试验 > DNA结构和改变
作者: Antonio Sarno
Antonio SarnoAffiliation: Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway
For correspondence: antonio.sarno@ntnu.no
Bio-protocol author page: a789
Vol 3, Iss 17, 9/5/2013, 2895 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.878

[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).

[Abstract]

Materials and Reagents

  1. NH4HCO3 (reagent grade ≥ 99% purity)
  2. MgCl2 (reagent grade ≥ 99% purity)
  3. CaCl2 (reagent grade ≥ 99% purity)
  4. DNase I from bovine pancreas (F. Hoffmann-La Roche, catalog number: 04716728001 )
  5. Nuclease P1 from P. citrinum (Sigma-Aldrich, catalog number: N8630-1VL )
  6. Phosphodiesterase I from C. adamanteus venom (Sigma-Aldrich, catalog number: P3242-1VL )
  7. Alkaline phosphatase from E. coli (Sigma-Aldrich, catalog number: P5931-100UN )
  8. 8-hydroxy-2’-deoxyguanosine (8-oxo-dGuo) (Sigma-Aldrich, catalog number: H5653-1MG )
  9. [15N5]-8-hydroxy-2’-deoxyguanosine (Cambridge Isotope Laboratories, catalog number: NLM-6715-0 ) (this is the internal standard)
  10. DNeasy® Blood & Tissue Kit (QIAGEN, catalog number: 69506 )
  11. LC/MS-grade methanol
  12. Hydrolysis buffer (see Recipes)
  13. Solvent A (see Recipes)
  14. Solvent B (see Recipes)

Equipment

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

Procedure

  1. DNA isolation
    DNA can be isolated using a variety of methods. We used the DNeasy® Blood Tissue Kit from QIAGEN, but other analogous kits are likely to yield similar results so long as they are not phenol based. Phenol-based DNA isolation has been shown to oxidize DNA in vitro and therefore overestimate 8-oxo-dGuo (Hamilton et al., 2001). Moreover, the final DNA elution step should be performed with water because most elution buffers contain EDTA, which inhibit nucleases.
  2. Enzymatic hydrolysis of DNA
    1. In 40 μl hydrolysis buffer, add
      1. 1 U DNase I
      2. 0.2 mU phosphodiesterase I
      3. 0.1 U alkaline phosphatase
      4. 1.25 pmol [15N5]-8-hydroxy-2’-deoxyguanosine internal standard
        (this gives a final concentration of 10 nM on the LC/MS/MS)
      5. 0.5-5 μg DNA
    2. Incubate at 37 °C for 6 h to overnight.
    3. Add five volumes of ice-cold methanol to the samples and mix well by vortexing. This step precipitates enzymes and salts prior so that (1) they don’t interfere with analyte ionization and (2) they don’t precipitate upon exposure to organic mobile phase during chromatography, which can cause clogs.
    4. Centrifuge samples at 16,000 x g for 20 min at 4 °C.
    5. Transfer the supernatant to new tubes. The pellet contains precipitated enzymes and salts that can interfere with analysis and can be discarded.
    6. Vacuum centrifuge the supernatant until dry.
    7. Dissolve the resulting residue in 25 μl 5% methanol in water.
    8. Inject 20 μl sample into the LC/MS/MS for analysis.
  3. A standard curve was made in 5% methanol in water containing 0.1-500 nM 8-oxo-dGuo, containing 10 nM internal standard.
  4. HPLC program (flow rate 300 μl/min)
    1. 5% solvent A for 0.5 min.
    2. Ramp to 90% solvent B over 6 min.
    3. Hold at 90% solvent B for 1.5 min.
    4. Re-equilibrate with 5% solvent A for 5 min.
  5. MS/MS program
    1. MS/MS acquisition should be used with positive electrospray ionization in multiple reaction monitoring mode.
      Note: The mass spectrometer settings are instrument specific and are therefore not included in this protocol.
    2. Mass transition for 8-hydroxy-2’-deoxyguanosine: 284.1 -> 168.2.
    3. Mass transition for [15N5]-8-hydroxy-2’-deoxyguanosine: 289.2 -> 173.1.
  6. Quantification
    1. Integrate the area under the peaks.
    2. Divide the 8-oxo-dGuo peak area by the internal standard peak area for all samples.
    3. Make a standard curve from the standards of known concentrations.
    4. Use the slope and y-intercept from the standard curve to calculate the concentration of the unknowns.
  7. (Optional) Deoxynucleoside quantification
    1. Dilute 1 μl of sample prior to MS injection 1:1,000 in 5% methanol in water. The reason for the dilution is that the canonical deoxynucleosides are much more abundant than 8-oxo-dGuo and would saturate the mass spectrometer’s detector if injected undiluted.
    2. Inject 20 μl of diluted sample for LC/MS/MS analysis of deoxynucleosides.
    3. Use the same HPLC program as for 8-oxo-dGuo.
    4. Mass transitions for deoxyguanosine, deoxycytidine, deoxyadenosine, and thymidine: 252.1 ->136.1, 228.1 -> 112.1, 268.1 -> 152.0, and 243.1 -> 127.0, respectively.
    5. Use standard curves to quantify deoxynucleoside concentrations.
    6. Use the following formula to calculate 8-oxo-dGuo per 106 nucleosides:
      (mol 8-oxo-dGuo/[mol dAdo + dGuo + dCyd + Thd]) x 1,000,000

Recipes

  1. Hydrolysis buffer
    100 mM NH4HCO3 (pH 7.6)
    10 mM MgCl2
    1 mM CaCl2
  2. Solvent A
    0.1% formic acid in water
  3. Solvent B
    0.1% formic acid in methanol

Notes

  1. Hydrolysis procedure. The hydrolysis procedure used here is one of many viable alternatives. When choosing a hydrolysis method for nucleoside analysis one must consider the following:
    1. Does the method affect the bases? DNA can be chemically hydrolyzed, but this is more risky because the bases themselves are subject to damage. Some enzymes also have unintended activity. For example, commercial alkaline phosphatase has been shown to contain deaminase activity (or contamination by deaminases) (Dong and Dedon, 2006; Dong et al., 2003).
    2. How important is a short hydrolysis reaction time? Some nucleoside modifications can arise spontaneously in water and a short hydrolysis reaction time is therefore worth the extra cost and effort necessary. Our group has also measured genomic uracil, which can arise spontaneously from cytosine deamination in water. We therefore developed a method to hydrolyze DNA in 50 min instead of 6 h (Galashevskaya et al., 2013). Adding even more enzymes, one can lower the reaction time to 15-30 min at room temperature (using DNase I, SVPD, micrococcal nuclease, omnicleave, benzonase, alkaline phosphatase, and Antarctic phosphatase; unpublished results by Sarno, 2013). Note that adding more enzymes significantly increases the reaction cost.
  2. Cleanliness. Mass spectrometry is a very sensitive technique, so great care should be taken to maintain a clean laboratory environment. Depending on the instrument and reagent quality, the assay can detect down to 0.1-0.5 fmol analyte. Thus, always ensure that all equipment and surfaces are clean and autoclaved if possible (e.g. pipettes, tips, tubes, centrifuges, etc.). Note that dust collects on surfaces over time, so even though a laboratory space may be contaminated even though it has not been used for some time. It is usually enough to wipe equipment and surfaces down with a laboratory wipe and deionized or milliQ water followed by either ethanol or isopropanol.
  3. Yield. We have performed the assay with 0.5-5 μg DNA and have always measured 8-oxo-dGuo above the assay’s limit of quantification. Nevertheless, one should always attempt to use as much DNA as possible (up to 5 μg) to ensure that there is enough measureable 8-oxo-dGuo. Regarding DNA yield: We have obtained an average of ~3 μg DNA per 106 cells from a multiple myeloma cell line using the DNeasy kit.
  4. Replicates. One should optimally have three technical replicates per sample. Thus, when analyzing 5 μg DNA, one should have at least 15 μg for three runs of 5 μg each. Additionally, one should always perform three independent experiments. Thus, one should have 3 x 15 μg per result.
  5. Quantification. Although it is possible to normalize the amount of 8-oxo-dGuo measured to μg DNA used in the initial hydrolysis reaction, it is more accurate and reproducible to compare 8-oxo-dGuo per (106) deoxynucleoside. This involves a single additional step and no extra material.


    Figure 1. Typical chromatograms in 8-oxo-dGuo analysis. A. 50 nM 8-oxo-dGuo and 10 nM internal standard dissolved in 5% methanol in water. B. 8-oxo-dGuo from 5 μg commercially obtained salmon sperm DNA containing 10 nM internal standard.
    Note that the peaks that don’t co-elute with the internal standard are discarded as contaminants.


    Figure 2. Visualized summary of the method.

Acknowledgments

This protocol is adapted from Sousa et al. (2013).

References

  1. Delaney, S., Jarem, D. A., Volle, C. B. and Yennie, C. J. (2012). Chemical and biological consequences of oxidatively damaged guanine in DNA. Free Radic Res 46(4): 420-441.
  2. Dong, M., Wang, C., Deen, W. M. and Dedon, P. C. (2003). Absence of 2'-deoxyoxanosine and presence of abasic sites in DNA exposed to nitric oxide at controlled physiological concentrations. Chem Res Toxicol 16(9): 1044-1055.
  3. Dong, M. and Dedon, P. C. (2006). Relatively small increases in the steady-state levels of nucleobase deamination products in DNA from human TK6 cells exposed to toxic levels of nitric oxide. Chem Res Toxicol 19(1): 50-57.
  4. Galashevskaya, A., Sarno, A., Vagbo, C. B., Aas, P. A., Hagen, L., Slupphaug, G. and Krokan, H. E. (2013). A robust, sensitive assay for genomic uracil determination by LC/MS/MS reveals lower levels than previously reported. DNA Repair (Amst) 12(9): 699-706. 
  5. Hamilton, M. L., Guo, Z., Fuller, C. D., Van Remmen, H., Ward, W. F., Austad, S. N., Troyer, D. A., Thompson, I. and Richardson, A. (2001). 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 29(10): 2117-2126.
  6. Sousa, M. M., Zub, K. A., Aas, P. A., Hanssen-Bauer, A., Demirovic, A., Sarno, A., Tian, E., Liabakk, N. B. and Slupphaug, G. (2013). An inverse switch in DNA base excision and strand break repair contributes to melphalan resistance in multiple myeloma cells. PLoS One 8(2): e55493. 

材料和试剂

  1. NH 4 HCO 3(试剂级≥99%纯度)
  2. MgCl 2(试剂级≥99%纯度)
  3. CaCl 2(试剂级≥99%纯度)
  4. 来自牛胰腺的DNA酶I(F.Hoffmann-La Roche,目录号:04716728001)
  5. 来自p的核酸酶P1。 柠檬苦素(Sigma-Aldrich,目录号:N8630-1VL)
  6. 磷酸二酯酶I。 adamanteus毒液(Sigma-Aldrich,目录号:P3242-1VL)
  7. 来自E的碱性磷酸酶。 大肠杆菌(Sigma-Aldrich,目录号:P5931-100UN)
  8. 8-羟基-2'-脱氧鸟苷(8-氧代-dGuo)(Sigma-Aldrich,目录号:H5653-1MG)
  9. [剑桥同位素实验室(Cambridge Isotope Laboratories),目录号:NLM-6715-0)(这是内部标准) br />
  10. DNeasy ® Blood& Tissue Kit(QIAGEN,目录号:69506)
  11. LC/MS级甲醇
  12. 水解缓冲液(参见配方)
  13. 溶剂A(参见配方)
  14. 溶剂B(参见配方)

设备

  1. Vortexer
  2. 微量离心机
  3. 真空离心机
  4. LC/MS/MS:我们使用耦合到API 5000三重四极杆质谱仪(Applied Biosystems)的LC-20AD HPLC系统(Shimadzu Corporation)
  5. Zorbax SB-C18反相色谱柱(2.1×150mm,i.d.,3.5μm)(Agilent Technologies)

程序

  1. DNA分离
    可以使用多种方法分离DNA。 我们使用QIAGEN的DNeasy Blood Tissue Kit,但是其他类似的试剂盒可能产生类似的结果,只要它们不是基于酚的。 基于酚的DNA分离已经显示在体外氧化DNA,因此高估了8-氧代-dGuo(Hamilton等人,2001)。 此外,最后的DNA洗脱步骤应该用水进行,因为大多数洗脱缓冲液含有抑制核酸酶的EDTA
  2. DNA的酶水解
    1. 在40μl水解缓冲液中,加入
      1. 1 U DNase I
      2. 0.2 mU磷酸二酯酶I
      3. 0.1 U碱性磷酸酶
      4. 1.25pmol [15 H] N 5 - ] - 8-羟基-2'-脱氧鸟苷内标
        (这使得在LC/MS/MS上的最终浓度为10nM)
      5. 0.5-5μgDNA
    2. 在37℃孵育6小时至过夜
    3. 向样品中加入5体积的冰冷甲醇,并通过涡旋混合均匀。 这一步骤沉淀酶和盐,以便(1)它们不干扰分析物电离,和(2)在色谱过程中暴露于有机流动相时它们不沉淀,这可能引起堵塞。
    4. 在4℃下以16,000×g离心样品20分钟
    5. 转移上清液到新管。 沉淀含有沉淀的酶和盐,可以干扰分析,可以丢弃
    6. 真空离心上清液至干
    7. 将所得残留物溶于25μl5%甲醇的水溶液中
    8. 将20μl样品注入LC/MS/MS进行分析
  3. 在含有0.1-500nM 8-氧代-dGuo的含有10nM内标的5%甲醇水溶液中制备标准曲线。
  4. HPLC程序(流速300μl/min)
    1. 5%溶剂A洗脱0.5分钟
    2. 在6分钟内,升至90%溶剂B.
    3. 在90%溶剂B保持1.5分钟。
    4. 用5%溶剂A重新平衡5分钟
  5. MS/MS程序
    1. MS/MS采集应在多反应监测模式下使用正电喷雾离子化。
      注意:质谱仪设置是仪器特定的,因此不包括在此协议中。
    2. 8-羟基-2'-脱氧鸟苷的质量转变:284.1 - 168.2。
    3. [15S] -N5S] -8-羟基-2'-脱氧鸟苷的质量转变:289.2→[
  6. 定量
    1. 集成峰下面积。
    2. 将8-氧代-dGuo峰面积除以所有样品的内标峰面积。
    3. 从已知浓度的标准品制作标准曲线。
    4. 使用来自标准曲线的斜率和y截距来计算未知数的浓度
  7. (可选)脱氧核苷定量
    1. 在MS注射前稀释1μl样品,在5%甲醇的水中1:1000。 稀释的原因是典型的脱氧核苷比8-氧代-dGuo丰富得多,如果未稀释的话,质谱仪的检测器将饱和。
    2. 注入20μl稀释的样品用于脱氧核苷的LC/MS/MS分析
    3. 使用与8-氧代-dGuo相同的HPLC程序
    4. 脱氧鸟苷,脱氧胞苷,脱氧腺苷和胸苷的质量跃迁:252.1-> 136.1,228.1-> 112.1,268.1 - > 152.0和243.1 - > 127.0。
    5. 使用标准曲线来定量脱氧核苷浓度
    6. 使用以下公式计算每10 6个核苷的8-氧代-dGuo:
      (mol 8-氧代-dGuo/[mol dAdo + dGuo + dCyd + Thd])×1,000,000

食谱

  1. 水解缓冲液
    100mM NH 4 HCO 3(pH 7.6)
    10mM MgCl 2/
    1mM CaCl 2
  2. 溶剂A
    0.1%甲酸水溶液
  3. 溶剂B
    0.1%甲酸的甲醇溶液

笔记

  1. 水解程序。 这里使用的水解方法是许多可行的替代方案之一。 当选择核苷分析的水解方法时,必须考虑以下因素:
    1. 方法是否影响碱基? DNA可以被化学水解,但是这是更危险的,因为碱基本身受到损伤。一些酶也具有非预期的活性。例如,商业碱性磷酸酶已显示含有脱氨酶活性(或脱氨酶污染)(Dong和Dedon,2006; Dong等人,2003)。
    2. 短的水解反应时间有多重要?一些核苷修饰可以在水中自发产生,因此短的水解反应时间值得额外的成本和所需的努力。我们的组还测量了基因组尿嘧啶,其可以自发地从水中的胞嘧啶脱氨产生。因此,我们开发了一种在50分钟而不是6小时内水解DNA的方法(Galashevskaya等人,2013)。添加甚至更多的酶,可以在室温下(使用DNase I,SVPD,微球菌核酸酶,omnicleave,benzonase,碱性磷酸酶和南极磷酸酶,Sarno,2013的未公开的结果)将反应时间降低至15-30分钟。注意,添加更多的酶会显着增加反应成本
  2. 清洁。质谱是一种非常敏感的技术,因此应当非常小心保持清洁的实验室环境。根据仪器和试剂质量,测定可以检测低至0.1-0.5 fmol分析物。因此,如果可能,请始终确保所有设备和表面都干净,并进行高压灭菌(例如移液器,提示,管,离心机,等)。注意,随着时间的推移,灰尘聚集在表面上,因此即使实验室空间可能被污染,即使它已经使用了一段时间。通常使用实验室擦拭物和去离子水或milliQ水,然后用乙醇或异丙醇擦拭设备和表面即可。
  3. 产量。我们已经用0.5-5μgDNA进行了测定,并且总是测量8-氧代-dGuo高于测定的定量极限。然而,应该总是尽量使用尽可能多的DNA(高达5μg),以确保有足够的可测量的8-氧代-dGuo。关于DNA产量:我们使用DNeasy试剂盒从多发性骨髓瘤细胞系获得了每10 6个细胞〜3μgDNA的平均值。
  4. 复制。每个样品最好具有三个技术重复。因此,当分析5μgDNA时,对于每次5μg的三次运行,应当具有至少15μg。另外,应该总是执行三个独立实验。因此,每个结果应该有3 x 15μg。
  5. 定量。虽然可以将在初始水解反应中使用的测定为μgDNA的8-氧代-dGuO的量标准化,但是比较8-氧代-dGuo /(10 6)脱氧核苷。这涉及一个额外的步骤,没有额外的材料

    图1.8-氧代-dGuo分析中的典型色谱图A.溶于5%甲醇的水中的50nM 8-氧代-dGuo和10nM内标。来自5μg商业获得的含有10nM内标的鲑鱼精DNA的B8-氧代-dGuo。
    请注意,不与内标共流出的峰会作为污染物丢弃。


    图2.方法的可视化摘要。

致谢

该协议改编自Sousa等人。(2013)。

参考文献

  1. Delaney,S.,Jarem,D.A.,Volle,C.B.and Yennie,C.J。(2012)。 DNA中氧化损伤的鸟嘌呤的化学和生物学后果。自由Rad Res 46(4):420-441。
  2. Dong,M.,Wang,C.,Deen,W.M.and Dedon,P.C。(2003)。 缺乏2'-脱氧呋喃核苷以及在受控生理浓度下暴露于一氧化氮的DNA中存在脱碱基位点 。 Chem Res Toxicol 16(9):1044-1055。
  3. Dong,M。和Dedon,P.C。(2006)。 暴露于人类TK6细胞的DNA中核碱基脱氨基产物的稳态水平相对较小的增加毒性水平的一氧化氮。 Chem Res Toxicol 19(1):50-57。
  4. Galashevskaya,A.,Sarno,A.,Vagbo,C.B.,Aas,P.A.,Hagen,L.,Slupphaug,G.and Krokan,H.E。 通过LC/MS/MS测定基因组尿嘧啶的稳定,灵敏的测定显示,比以前报道的水平更低。 DNA Repair(Amst) 12(9):699-706。 
  5. Hamilton,M.L.,Guo,Z.,Fuller,C.D.,Van Remmen,H.,Ward,W.F.,Austad,S.N.,Troyer,D.A.,Thompson,I.and Richardson,A。(2001)。 使用碘化钠可靠地评估核和线粒体DNA中的8-氧代-2-脱氧鸟苷水平方法分离DNA。核酸研究 29(10):2117-2126。
  6. Sousa,M.M.,Zub,K.A.,Aas,P.A.,Hanssen-Bauer,A.,Demirovic,A.,Sarno,A.,Tian,E.,Liabakk,N.B.and Slupphaug,G。(2013)。 DNA碱基切除和链断裂修复中的反向切换有助于多发性骨髓瘤细胞中的美法仑抗性。/a> PLoS One 8(2):e55493。
English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


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



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook