Name: hplc measurement of the dna oxidation biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in cultured cells and animal tissues
Uploaded: 2015-08-01T14:00:00
Description: Watch this Scientific Journal Video about HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2Ã¢â‚¬â„¢-deoxyguanosine, in Cultured Cells and Animal Tissues at JoVE.com

该研究是由加拿大卫生部基因组学研究和发展倡议（GRDI）和加拿大监管策略生物技术（华润雪花）。作者有没有利益冲突。

确保所有牧，住房，处理和实验遵守当地的法律和法规，而且实验的协议之前，开始任何研究批准。对于所描述的实验中，动物护理，处理和治疗批准了加拿大卫生署动物管理委员会。见“试剂表”，供应商的信息。 1.采集生物样品<ol><li>细胞或动物组织<ol><li>生长在含有10％胎牛血清，青霉素100单位/ ml和链霉素100μg/ ml的F12-K媒体人肺泡腺癌A549细胞。 </li><li>种子细胞在每一...

<ol>
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虽然-8-氧代dGuo已被报告为脱氧核糖核酸氧化的有用的生物标志物，其可靠的量化可以构成挑战。虽然有几种公开的方法存在，有必要对协议的全面的，描述概述，以允许研究人员部署在他们的实验室的方法。这里，我们提出的HPLC为基础的协议，将允许新用户建立一种有效的方法-8-氧代dGuo检测和定量的详细概述。 已经描述了8个氧代dGuo量化三种主要的方法。这些包括酶联免疫...

活性氧（ROS），其稳态水平期间许多病理学病症和xenotoxic代谢能增加，有助于氧化性DNA损伤的频率增加。在几个可能的核碱基的氧化产物，氧化性DNA损伤可容易地使用稳定标记测量-8-氧代-7,8-二氢-2&#39;-脱氧鸟苷（8-氧dGuo），它是2的氧化形式之一&#39; -deoxyguanosine（dGuo）1。 -8-氧代dGuo是最丰富的DNA损伤2，因此，已研究以更详细的DNA氧化标志物尽管多种DNA氧化产物3的存在。在人类中，这种损伤可以通过碱基切除修复了8羟基鸟嘌呤糖苷酶1（hOGG1基因）4修复。如果左未修理，8-氧dGuo可以向形成的碱基对替代突变（ 即 ，G到T颠换）4。重要的是，8-氧dGuo是一个既定的标记FO- [R DNA损伤有关的启动和促进癌变2。因此，8-氧代- dGuo准确定量是氧化性DNA损伤5的有用和理想的生物标志物。 人们普遍混乱关于对氧化损伤的形式的2-脱氧鸟苷，此外，化合物（S）的正确名称常规测量作为氧化性DNA损伤6的生物标志物的正确名称的文献。的8-氧dGuo（ 图1中示出）的6,8-二酮和6-烯醇，8-酮互变异构形式都在文献5,7中讨论的两个最突出的互变异构体。的6,8-二酮的形式是在7.4的生理pH值的最突出的形式，并且是最突出的DNA氧化产物7。因此，8-氧dGuo，而不是8羟基dGuo是用于该氧化产物6的最适当的名称。同样重要的是要注意，2-脱氧鸟苷（dGuo），而不是nucleob酶鸟嘌呤（卦）或核糖鸟苷（国），分别是由大多数方法6检测。 精确检测和8-氧代dGuo量化是具有挑战性的，原因是：在该DNA样品的消化ⅰ）变异，ⅱ）dGuo的不定氧化成8-氧dGuo可能发生样品制备过程中，和iii）需要在分析HPLC-ED法8验证有效。在这个协议中，我们通过提供条件，有利的完整的DNA消化和ii）通过包含金属螯合剂和螯合剂处理的溶液和一种特殊的DNA分离试剂，而ⅲ）仅部分通过包含寻址旨在实现ⅰ）阳性对照，从而提供了该方法能够检测-8-氧代dGuo生物样品中。进一步验证超出了本文的范围。但是，我们相信，这一协议将有助于准用户确定在何种程度上他们需要正式验证协议，这取决于它们的目的。所需的方法的正式验证步骤的清单还设置。在8氧dGuo检测方法的开发和部署，公布的方法进行了回顾和巩固。因此，该方法消除了需要从几个公布来源往往缺乏重要的实验细节，同时还提供测试快速和简单的方式，如果该方法用于-8-氧代dGuo的检测和定量已成功地采用了收集信息。这种适应方法采用成功地分析DNA样本培养细胞和小鼠组织。这个视频文章将有助于其他群体建立一种有效的方法可靠的检测和8-氧dGuo定量用HPLC-ED。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>8-oxo-dGuo standard</td>
			<td>Cayman Chemical Company</td>
			<td>89320</td>
			<td>Inappropriately referred to as &#34;8-hydroxy-2&#39;-deoxy Guanosine&#34; - see Fig. 1 and text for details</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P5931</td>
			<td>From E.coli</td>
		</tr>
		<tr>
			<td>Chelex 100</td>
			<td>Sigma-Aldrich</td>
			<td>C7901</td>
			<td>Chelates heavy metals</td>
		</tr>
		<tr>
			<td>Desferoxamine mesylate</td>
			<td>Sigma-Aldrich</td>
			<td>D9533</td>
			<td></td>
		</tr>
		<tr>
			<td>dGuo standard</td>
			<td>Sigma-Aldrich</td>
			<td>D7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibasic sodium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>S9390</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA from salmon sperm</td>
			<td>Sigma-Aldrich</td>
			<td>D1626</td>
			<td>Sodium salt</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td>D4527</td>
			<td>TypeII, from bovine pancreas</td>
		</tr>
		<tr>
			<td>DNAzol</td>
			<td>Invitrogen</td>
			<td>10503-27</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E4884</td>
			<td>The compound would not completely dissolve until solution pH is adjusted to 8.0 with e.g. NaOH</td>
		</tr>
		<tr>
			<td>F12-K media</td>
			<td>ATCC</td>
			<td>30-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>ATCC</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Guard column</td>
			<td>Chromatographic Specialties</td>
			<td>YBA 99S03 0204GC</td>
			<td>Protects colum from contamination; may also lead to pressure build-up</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Monobasic sodium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Invitrogen</td>
			<td>15190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphodiesterase I enzyme&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P3243</td>
			<td>Type II from Crotalus adamaneus venom</td>
		</tr>
		<tr>
			<td>Teflon homogenizer</td>
			<td>Thomas Scientific</td>
			<td>7724T-1 or 7724T-5 for 1 or 5 mL, respectively</td>
			<td>Volume (holding capacity) depends on the amount of sample to be processed.</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Invitrogen</td>
			<td>15050-065</td>
			<td></td>
		</tr>
		<tr>
			<td>YMC-BASIC column with bonded spherical silica</td>
			<td>Chromatographic Specialties</td>
			<td>YBA 99S03 1546WT</td>
			<td></td>
		</tr>
	</tbody>
</table>

hplc measurement of the dna oxidation biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in cultured cells and animal tissues

氧化应激与许多生理和病理过程，以及生物外源性代谢相关，导致生物大分子的氧化，包括DNA。因此，高效的检测DNA氧化是多种学科的研究，包括药物和毒物的重要。氧化损伤的DNA的一个共同的生物标志物是-8-氧代-7,8-二氢-2&#39;-脱氧鸟苷（8-氧代dGuo;经常误称为8-羟基-2&#39;-脱氧鸟苷（8-OH-dGuo或8氧代dG的））。对于-8-氧代dGuo测量用高压液相色谱电化学检测器（HPLC-ED）几个协议已被描述。然而，这些主要适用于具有促氧化剂处理纯化DNA。此外，由于实验室之间方法上的差异，主要是由于在分析设备的差异，采用用于检测-8-氧代dGuo通过HPLC-ED公布的方法需要由每个实验室仔细优化。一个全面的协议，描述这样一个优化过程，是缺乏。这里，一个详细的协议是从培养的细胞或动物组织中所述的8-氧dGuo通过HPLC-ED的检测，在DNA中。它说明了如何DNA样品制备能够容易且快速地优化，以尽量减少可能发生的样品制备过程中不希望的DNA的氧化。这个协议显示了如何检测-8-氧代dGuo与氧化剂溴酸钾3处理培养的人肺泡腺癌细胞（ 即 ，A549细胞），以及从暴露在多环芳烃二苯并（ 闪避，p）的屈小鼠的脾脏（DBC，前身为二苯（A，L）芘，DALP）。总体来说，这一工作说明了如何用HPLC-ED方法可以用于-8-氧代dGuo生物样品中的检测可以容易地优化。

dGuo观察到有4.7分钟而-8-氧代dGuo具有约6.4分钟（ 图2A和B）的保留时间，保留时间。有一个在两种分析物之间的峰高约1000倍的差异，如在图2C中看到的。伏安-8-氧代dGuo和dGuo通过运行标准在+0.2的范围内的工作电位，获得至1.1 V的最佳工作电位为-8-氧代dGuo被确定为+ 0.5V，和0.9 v对于dGuo（ 图3）。这些电位是与文献14,15中所述的其它玻璃碳电极?...

Watch this Scientific Journal Video about HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2Ã¢â‚¬â„¢-deoxyguanosine, in Cultured Cells and Animal Tissues at JoVE.com

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2ÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢-deoxyguanosine, in Cultured Cells and Animal Tissues

该协议的目的是将DNA氧化标志物的检测，8-氧代-7,8-二氢-2&#39;-脱氧鸟苷（8-氧dGuo）通过HPLC-ED，从培养的细胞或动物组织的DNA。

该DNA氧化标志物的高效液相色谱法测量，8-氧代-7,8-二氢-2&#39;-脱氧鸟苷，在培养细胞和动物组织

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

Research

JoVE Journal

Chemistry

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

稳态，稳定前的状态，单营业额动力学测量DNA糖基化酶活性

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

科研

化学

Folding and Characterization of a Bio-responsive Robot from DNA Origami

折叠的DNA折纸一个生物反应机器人与表征

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Construction of Models for Nondestructive Prediction of Ingredient Contents in Blueberries by Near-infrared Spectroscopy Based on HPLC Measurements

基于HPLC测量通过近红外光谱对在蓝莓成分的含量无损预测模型的构建

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

HPLC-基于化验，以监测外核苷酸/核苷代谢的人类慢性淋巴细胞白血病细胞

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Determining the Chemical Composition of Corrosion Inhibitor/Metal Interfaces with XPS: Minimizing Post Immersion Oxidation

确定缓蚀剂/金属界面的化学成分与XPS：最小化后浸入氧化

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, ...

Fabrication and Testing of Photonic Thermometers

光子温度计的制作与测试

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

同时测量 HDAC1 和 HDAC6 活动在 HeLa 细胞使用 UHPLC 女士

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

解析高强共聚物芳纶纤维使其力学性能的测定

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...