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

Denne forskning blev finansieret af Health Canada Genomics Research and Development Initiative (GRDI) og den canadiske Regulatory strategi for bioteknologi (CRSB). Forfatterne har ingen interessekonflikter.

Sikre, at alle dyrehold, bolig, håndtering og eksperimentering overholde lokale regler og bestemmelser, og at eksperimenter protokoller er godkendt forud for påbegyndes undersøgelsen. For de beskrevne eksperimenter blev dyr pleje, håndtering og behandling er godkendt af Health Canada Animal Care udvalget. Se &quot;Reagenser tabellen&quot; for leverandørernes oplysninger. 1. Indsamling Biologiske prøver <ol><li> Celler eller animalske væv <ol><li> Grow humane alveolære adenocarcinom...

<ol>
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L., Douki, T., Duez, P., Gremaud, E., Herbert, K., Hofer, T., Lasserre, L., Saint-Pierre, C., Favier, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+background+level+of+8-oxo-7,8-dihydro-2'-deoxyguanosine:+an+isotope+based+method+to+evaluate+artefactual+oxidation+of+DNA+during+its+extraction+and+subsequent+work-up.">Cellular background level of 8-oxo-7,8-dihydro-2'-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up.</a> Carcinogenesis. 23 (11), 1911-1918 (2002).</li><li>Gossen, J. A., De Leeuw, W. J. F., Tan, C. H. T., Zwarthoff, E. C., Berends, F., Lohman, P. H. M., Knook, D. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+8-oxoguanine+in+DNA+by+gas+chromatography-mass+spectrometry+and+HPLC-electrochemical+detection:+overestimation+of+the+background+level+of+the+oxidized+base+by+the+gas+chromatography-mass+spectrometry+assay.">Determination of 8-oxoguanine in DNA by gas chromatography-mass spectrometry and HPLC-electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography-mass spectrometry assay.</a> Chem. Res. 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Selvom 8-oxo-dGuo er blevet rapporteret som en nyttig biomarkør for DNA oxidation, kan dens pålidelig kvantificering en udfordring. Selvom der findes adskillige offentliggjorte metoder, er der behov for en omfattende, beskrivende oversigt over protokol til at tillade forskere at implementere metoden i deres laboratorier. Her præsenterer vi en detaljeret oversigt over en HPLC-baseret protokol, der vil tillade nye brugere til at etablere en effektiv metode til 8-oxo-dGuo detektion og kvantificering. <p class="jove_...

Reaktive ilt arter (ROS), hvis steady state niveauer kan stige under mange patologiske tilstande og xenotoxic stofskifte, bidrager til en øget frekvens af oxidative DNA-skader. Blandt flere mulige nukleobaser oxidationsprodukter kan let måles, oxidativ DNA-beskadigelse bruge den stabile markør 8-oxo-7,8-dihydro-2&#39;-deoxyguanosin (8-oxo-dGuo), som er en af ​​de oxiderede former af 2 &#39; -deoxyguanosine (dGuo) 1. 8-oxo-dGuo er den mest udbredte DNA læsion 2, og derfor er blevet studeret til nærmere som en DNA oxidation biomarkør trods eksistensen af multiple DNA oxidationsprodukter 3. Hos mennesker kan denne skade repareres via basen excision reparation ved 8-oxoguanine glycosylase 1 (hOGG1) 4. Hvis ikke repareres, kan 8-oxo-dGuo bidrager til dannelsen af basepar-substitutionsmutationer (dvs. G til T transversioner) 4. Vigtigere, 8-oxo-dGuo er en etableret markør for DNA-skader i forhold til initiering og fremme af carcinogenese 2. Derfor nøjagtig kvantificering af 8-oxo-dGuo er en nyttig og ønskelig biomarkør for oxidativ DNA-skade 5. Der er udbredt forvirring i litteraturen vedrørende de korrekte navne for oxidativt-beskadigede former af 2-deoxyguanosin og desuden det korrekte navn for forbindelsen (r) rutinemæssigt målt som biomarkør for oxidativ DNA-skade 6. De 6,8-diketo og 6-enol, 8-keto tautomere former af 8-oxo-dGuo (vist i figur 1) er de to mest fremtrædende tautomerer omtales i litteraturen 5,7. Den 6,8-diketo formular er den mest fremtrædende form ved fysiologisk pH på 7,4, og er den mest fremtrædende DNA oxidationsprodukt 7. Derfor, 8-oxo-dGuo, snarere end 8-hydroxy-dGuo er den mest passende navn for dette oxidationsprodukt 6. Det er også vigtigt at bemærke, at 2-deoxyguanosin (dGuo), snarere end nucleobase guanin (Gua) eller ribonucleosid guanosin (Guo) henholdsvis detekteres ved de fleste metoder 6. Nøjagtig påvisning og kvantificering af 8-oxo-dGuo er udfordrende på grund af: i) variation i fordøjelsen af DNA-prøven, ii) utilsigtet oxidation af dGuo til 8-oxo-dGuo som kan forekomme under prøveforberedelse, og iii) behovet for effektiv validering af den analytiske HPLC-ED-metoden 8. I denne protokol, vi sigter mod at opnå i) ved at skabe vilkår, gunstige for komplet DNA fordøjelse og ii) ved inddragelse metalchelatoren og chelator-behandlede løsninger og en særlig DNA-isolerende reagens, mens iii) blev kun delvist behandles i inddragelse af positive kontroller og således tilvejebringer, at fremgangsmåden er i stand til at detektere 8-oxo-dGuo i biologiske prøver. Yderligere validering er uden for rammerne af dette papir. Men vi er overbeviste om, at denne protokol vil hjælpe den potentiellebrugere bestemme, i hvilket omfang de skal formelt godkende protokollen, afhængigt af deres formål. En liste over trin, der kræves for den formelle validere metoden tilvejebringes yderligere. Under udvikling og implementering af en metode til 8-oxo-dGuo afsløring, blev offentliggjort metoder revideret og konsolideret. Således er denne metode eliminerer behovet for at indsamle oplysninger fra flere offentliggjorte kilder, der ofte mangler vigtige eksperimentelle detaljer og giver samtidig hurtige og ligetil hjælp af test, hvis metode til påvisning og kvantificering af 8-oxo-dGuo er blevet vedtaget med succes. Denne tilpassede metode blev anvendt med succes analysere DNA-prøver fra dyrkede celler og murine væv. Denne video artikel vil hjælpe andre grupper med at etablere en effektiv metode til pålidelig detektion og kvantificering af 8-oxo-dGuo ved 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

Oxidativt stress er forbundet med mange fysiologiske og patologiske processer, samt xenobiotisk metabolisme, hvilket fører til oxidation af biomakromolekyler, herunder DNA. Derfor er det vigtigt for en række forsknings- discipliner, herunder medicin og toksikologi effektiv detektion af DNA oxidation. En fælles biomarkør for oxidativt beskadiget DNA er 8-oxo-7,8-dihydro-2&#39;-deoxyguanosin (8-oxo-dGuo, ofte fejlagtigt betegnet som 8-hydroxy-2&#39;-deoxyguanosin (8-OH-dGuo eller 8 -oxo-dG)). Adskillige protokoller til 8-oxo-dGuo måling ved højtryksvæskekromatografi med elektrokemisk detektion (HPLC-ED) er blevet beskrevet. Imidlertid blev disse hovedsagelig anvendes til oprenset DNA behandlet med pro-oxidanter. Hertil kommer, på grund af metodologiske forskelle mellem laboratorier, primært på grund af forskelle i analyseudstyr, vedtagelse af publicerede metoder til påvisning af 8-oxo-dGuo ved HPLC-ED kræver omhyggelig optimering af hvert laboratorium. Enomfattende protokol, som beskriver en sådan optimeringsproces, mangler. Her er en detaljeret protokol beskrevet for påvisning af 8-oxo-dGuo ved HPLC-DE, i DNA fra dyrkede celler eller dyrevæv. Det illustrerer, hvordan forberedelse DNA-prøve kan let og hurtigt optimeret til at minimere uønsket DNA oxidation, der kan forekomme under forberedelsen. Denne protokol viser, hvordan man kan opdage 8-oxo-dGuo i dyrkede humane alveolære adenocarcinomceller (dvs. A549-celler) behandlet med oxidationsmidlet KBrO 3, og fra milten af mus udsat for den polycykliske dibenzo aromatisk carbonhydrid (def, s) chrysen (DBC, tidligere kendt som dibenzo (a, l) pyren, DalP). Samlet set dette arbejde illustrerer, hvordan en HPLC-ED metode kan let optimeres til påvisning af 8-oxo-dGuo i biologiske prøver.

dGuo blev observeret at have en retentionstid på 4,7 min mens 8-oxo-dGuo havde en retentionstid på ca. 6,4 min (figur 2A og B). Der er omkring 1000 gange forskel i tophøjderne mellem de to analytter, som det ses i figur 2C. Voltammogrammer for 8-oxo-dGuo og dGuo blev opnået ved kørende standarder på en arbejdende potentiale i intervallet 0,2-1,1 V. Den optimale arbejder potentiale for 8-oxo-dGuo blev bestemt til at være +0,5 V og 0,9 V for dGuo (figur 3)....

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

Målet med denne protokol er påvisningen af ​​DNA oxidation markering, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosin (8-oxo-dGuo) ved HPLC-DE, i DNA fra dyrkede celler eller dyrevæv.

HPLC Måling af DNA Oxidation Biomarkør, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosin i dyrkede celler og animalsk væv

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

Steady-state, Pre-steady-state, og Single-omsætningen Kinetic måling for DNA glycosylase Activity

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

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

Foldning og karakterisering af en Bio-responsiv Robot fra DNA Origami

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

Konstruktion af Modeller for destruktiv Forudsigelse af indholdsstof Indhold i Blåbær ved Near-infrarød spektroskopi Baseret på HPLC Målinger

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-baserede analysen til overvågning Ekstracellulær Nukleotid / nukleosid Metabolisme i Human kronisk lymfatisk leukæmi celler

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

Syntese af Protein biokonjugater<em&gt; via</em&gt; Cystein-maleimid Kemi

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

Bestemmelse af kemiske sammensætning af korrosionsinhibitor / Metal Grænseflader med XPS: Minimering Indlæg Immersion Oxidation

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

Fabrikation og afprøvning af fotoniske termometre

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

Samtidig måling af HDAC1 og HDAC6 aktivitet i HeLa celler ved hjælp af UHPLC-MS

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

DNA-magnetiske partikel bindende analyse af dynamiske og elektroforese lysspredning

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

Udrede højstyrke Copolymer Aramid fibre til bestemmelse af mekaniske egenskaber

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