Esta pesquisa foi financiada pelo Canadá Health Initiative Genomics Research e Desenvolvimento (GRDI) ea Estratégia Regulatória Canadense de Biotecnologia (RCSB). Os autores não têm qualquer conflito de interesses.

<ol>
	<li>Helbock, H. J., Beckman, K. B., Shigenaga, M. K., Walter, P. B., Woodall, A. A., Yeo, H. C., Ames, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+oxidation+matters:+The+HPLC-electrochemical+assay+of+8-oxo-deoxyguianosine+and+8-oxo-guanine.">DNA oxidation matters: The HPLC-electrochemical assay of 8-oxo-deoxyguianosine and 8-oxo-guanine.</a> Proc. Natl. Acad. Sci. 95 (1), 288-293 (1998).</li><li>Valavanidis, A., Vlachogianni, T., Fiotakis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=8-hydroxy-2'+-deoxyguanosine+(8-OHdG):+A+critical+biomarker+of+oxidative+stress+and+carcinogenesis.">8-hydroxy-2' -deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis.</a> J. Environ. Sci Health C Environ. Carcinog. Ecotoxicol. Rev. 27 (2), 120-139 (2009).</li><li>Cadet, J., Bellon, S., Douki, T., Frelon, S., Gasparutto, D., Muller, E., Pouget, J. P., Ravanat, J. L., Romieu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+DNA+damage:+formation,+measurement,+and+biochemical+features.">Radiation-induced DNA damage: formation, measurement, and biochemical features.</a> J Environ Pathol Toxicol Oncol. 23 (1), 23-23 (2004).</li><li>Weiss, J. M., Goode, E. L., Ladiges, W. C., Ulrich, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymorphic+variation+in+hOGG1+and+risk+of+cancer:+a+review+of+the+functional+and+epidemiologic+literature.">Polymorphic variation in hOGG1 and risk of cancer: a review of the functional and epidemiologic literature.</a> Mol. Carcinog. 42 (3), 127-141 (2005).</li><li>Culp, S. J., Cho, B. P., Kadlubar, F. F., Evans, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+Conformational+Analyses+of+8-hydroxy-2'-deoxyguanosine.">Structural and Conformational Analyses of 8-hydroxy-2'-deoxyguanosine.</a> Chem. Res. Toxicol. 2 (6), 416-422 (1989).</li><li>Cooke, M. S., Loft, S., Olinski, R., Evans, M. D., Bialkowski, K., Wagner, J. R., Dedon, P. C., M&#248;ller, P., Greenberg, M. M., Cadet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+standardized+description+of+and+nomenclature+concerning+oxidatively+damaged+nucleobases+in+DNA.">Recommendations for standardized description of and nomenclature concerning oxidatively damaged nucleobases in DNA.</a> Chem. Res. Toxicol. 23 (4), 705-707 (2010).</li><li>Jang, Y. H., Goddard, W. A. 3. r. d., Noyes, K. T., Sowers, L. C., Hwang, S., Chung, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+principles+calculations+of+the+tautomers+and+pKa+values+of+8-oxoguanine:+implications+for+mutagenicity+and+repair.">First principles calculations of the tautomers and pKa values of 8-oxoguanine: implications for mutagenicity and repair.</a> Chem. Res. Toxicol. 15 (8), 1023-1035 (2002).</li><li>Park, J. -. H., Gopishetty, S., Szewczuk, L. M., Troxel, A. B., Harvey, R. G., Penning, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+8-oxo-7,8-dihydro-2'-deoxyguanosine+(8-oxo-dGuo)+by+PAH+o-quinones:+involvement+of+reactive+oxygen+species+and+copper(ii)/copper(i)+redox+cycling.">Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) by PAH o-quinones: involvement of reactive oxygen species and copper(ii)/copper(i) redox cycling.</a> Chem. Res. Toxicol. 18 (6), 1026-1037 (2005).</li><li>Mangal, D., Vudathala, D., Park, J. H., Lee, S. H., Penning, T. M., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+7,8-dihydro-8-oxo-2'-deoxyguanosine+in+cellular+DNA+during+oxidative+stress.">Analysis of 7,8-dihydro-8-oxo-2'-deoxyguanosine in cellular DNA during oxidative stress.</a> Chem. Res. Toxicol. 22 (5), 788-797 (2009).</li><li>Ravanat, J. 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. L., Vijg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+rescue+of+integrated+shuttle+vectors+from+transgenic+mice:+A+model+for+studying+mutations+in+vivo.">Efficient rescue of integrated shuttle vectors from transgenic mice: A model for studying mutations in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 86 (20), 7971-7975 (1989).</li><li>Van Campen, L. E., Murphy, W. J., Franks, J. R., Mathias, P. I., Toraason, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+DNA+damage+is+associated+with+intense+noise+exposure+in+the+rat.">Oxidative DNA damage is associated with intense noise exposure in the rat.</a> Hear Res. 164 (1-2), 164-161 (2002).</li><li>European Standards Committee on Oxidative DNA Damage (ESCODD). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+DNA+oxidation+in+human+cells+by+chromatographic+and+enzymic+methods.">Measurement of DNA oxidation in human cells by chromatographic and enzymic methods.</a> Free Radic. Biol. Med. 34 (8), 1089-1099 (2003).</li><li>Rebelo, I. A., Piedade, J. A., Oliveira-Brett, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+HPLC+method+with+electrochemical+detection+of+femtomoles+of+8-oxo-7,8-dihydroguanine+and+8-oxo-7,8-dihydro-2'-deoxyguanosine+in+the+presence+of+uric+acid.">Development of an HPLC method with electrochemical detection of femtomoles of 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of uric acid.</a> Talanta. 63 (2), 323-331 (2004).</li><li>Ravanat, J. -. L., Turesky, R. J., Gremaud, E., Trudel, L. J., Stadler, R. 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. Toxicol. 8 (8), 1039-1045 (1995).</li><li>Kawanishi, S., Murata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+DNA+damage+induced+by+bromate+differs+from+general+types+of+oxidative+stress.">Mechanism of DNA damage induced by bromate differs from general types of oxidative stress.</a> Toxicology. 221 (2-3), 172-178 (2006).</li><li>Tahara, S., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+mouse+splenic+cells+to+oxidative+DNA+damage+by+x-ray+irradiation.">Susceptibility of mouse splenic cells to oxidative DNA damage by x-ray irradiation.</a> Biol. Pharm. Bull. 27 (1), 105-108 (2004).</li><li>Garratt, L. W., Mistry, V., Singh, R., Sandhu, J. K., Sheil, B., Cooke, M. S., Sly, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+of+urinary+8-oxo-7,8-dihydro-2'-deoxyguanosine+is+adversely+affected+by+methodological+inaccuracies+when+using+a+commercial+ELISA.">Interpretation of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine is adversely affected by methodological inaccuracies when using a commercial ELISA.</a> Free Radic. Biol. Med. 48 (11), 1460-1464 (2012).</li><li>Cooke, M. S., Collins, A., Olinski, R., Rozalski, R., Loft, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harmonising+measurements+of+8-oxo-7,8-dihydro-2&#8242;-deoxyguanosine+in+cellular+DNA+and+urine.">Harmonising measurements of 8-oxo-7,8-dihydro-2&#8242;-deoxyguanosine in cellular DNA and urine.</a> Free Radic. Res. 46 (4), 541-553 (2012).</li><li>Cadet, J., Douki, T., Ravanat, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+oxidatively+generated+base+damage+in+cellular+DNA.">Measurement of oxidatively generated base damage in cellular DNA.</a> Mutat Res. 711 (1-2), 3-12 (2011).</li><li>Chomczynski, P., Mackey, K., Drews, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNAzol:+a+reagent+for+the+rapid+isolation+of+genomic+DNA.">DNAzol: a reagent for the rapid isolation of genomic DNA.</a> Biotechniques. 22 (3), 550-553 (1997).</li><li>Collins, A. R., Cadet, J., M&#246;ller, L., Poulsen, H. E., Vi&#241;a, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+we+sure+we+know+how+to+measure+8-oxo-7,8-dihydroguanine+in+DNA+from+human+cells.">Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells.</a> Arch Biochem Biophys. 423 (1), 57-65 (2004).</li><li>Badouard, C., M&#233;n&#233;zo, Y., Panteix, G., Ravanat, J. L., Douki, T., Cadet, J. <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+new+types+of+DNA+lesions+in+human+sperm.">Determination of new types of DNA lesions in human sperm.</a> Zygote. 16 (1), 9-13 (2008).</li><li>Cadet, J., Douki, T., Gasparutto, D., Ravanat, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+damage+to+DNA:+formation,+measurement+and+biochemical+features.">Oxidative damage to DNA: formation, measurement and biochemical features.</a> Mutat Res. 531 (1-2), 1-2 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Validation of analytical procedures: text and methodology Q2(R1). , (2015).</li></ol>

No conflict of interest or competing financial interests declared.

Apesar de 8-oxo-dGuo foi classificado como um biomarcador útil de oxidação do DNA, a sua quantificação confiável pode representar um desafio. Embora existam vários métodos publicados, há uma necessidade de uma visão abrangente, descritivo de protocolo para permitir que pesquisadores para implantar o método em seus laboratórios. Aqui apresentamos uma visão detalhada de um protocolo baseado em HPLC que irá permitir novos usuários para estabelecer um método eficaz para a detecção e quantificação de 8-ox...

Espécies reativas de oxigênio (ROS), cujos níveis de estado estacionário pode aumentar durante muitas condições patológicas e metabolismo xenotoxic, contribuir para um aumento da frequência de dano oxidativo ao DNA. Entre os vários possíveis nucleobases produtos de oxidação, lesões oxidativas do ADN pode ser facilmente medida utilizando o marcador estável de 8-oxo-7,8-di-hidro-2&#39;-desoxiguanosina (8-oxo-dGuo), que é uma das formas oxidadas de 2 &#39; -deoxyguanosine (dGuo) 1. 8-oxo-dGuo é o mais abundante lesão de DNA 2 e, por conseguinte, foi estudada a maior detalhe como um biomarcador oxidação DNA, apesar da existência de múltiplos produtos de oxidação de ADN 3. Nos seres humanos, este dano pode ser reparado através de base de reparo de excisão por 8-oxoguanine glicosilase 1 (hOGG1) 4. Se reparado, 8-oxo-dGuo pode contribuir para a formação da base de mutações de substituição de par (isto é, transversões de G para T) 4. Importante, 8-oxo-dGuo é um marcador estabelecido fodano do ADN r em relação à iniciação e promoção da carcinogénese 2. Portanto, a quantificação precisa de 8-oxo-dGuo é um biomarcador útil e desejável de dano oxidativo ADN 5. Há uma confusão generalizada na literatura sobre os nomes corretos para as formas oxidativamente-danificadas do 2-desoxiguanosina e, além disso, o nome correto do composto (s) medidos rotineiramente como um biomarcador de dano oxidativo DNA 6. Os 6,8-diceto-enol e 6, 8-ceto formas tautoméricas de 8-oxo-dGuo (mostrado na Figura 1) são os dois tautómeros mais proeminentes discutidos na literatura 5,7. A forma de ácido 6,8-diceto é a forma mais proeminente a pH fisiológico de 7,4, e é o mais proeminente do produto de oxidação de DNA 7. Portanto, 8-oxo-dGuo, em vez de 8-hidroxi-dGuo é o nome mais adequado para este produto de oxidação 6. É também importante notar que a 2-desoxiguanosina (dGuo), em vez de nucleobase guanina (Gua) ou guanosina ribonucleósido (Guo), respectivamente, é detectado pela maioria dos métodos 6. Detecção e quantificação de 8-oxo-dGuo preciso é um desafio devido a: i) a variabilidade na digestão da amostra de ADN, ii) oxidação acidental de dGuo a 8-oxo-dGuo que pode ocorrer durante a preparação da amostra, e iii) a necessidade eficaz para validação do método de HPLC analítico ED-8. Neste protocolo, com vista à consecução i) proporcionando condições favoráveis, para a digestão completa do ADN e ii) pela inclusão quelante de metal e soluções tratados com quelantes e um reagente especial de isolamento de DNA, enquanto iii) foi apenas parcialmente abordados pela inclusão de controlos positivos e proporcionando, assim, que o método é capaz de detectar 8-oxo-dGuo em amostras biológicas. Além disso validação está além do escopo deste artigo. No entanto, estamos confiantes de que este protocolo vai ajudar o prospectivoutilizadores determinar a extensão em que eles precisam validar formalmente o protocolo, dependendo das suas finalidades. Uma lista dos passos requeridos para a validação formal do método é ainda proporcionado. Durante o desenvolvimento e implantação de um método de detecção 8-oxo-dGuo, métodos publicados foram revistos e consolidados. Assim, este método elimina a necessidade de recolher informações a partir de várias fontes publicadas que carecem frequentemente importantes detalhes experimentais ao mesmo tempo proporcionar meios rápidos e simples de testar se o método para a detecção e quantificação de 8-oxo-dGuo foi adoptada com sucesso. Este método foi adaptado empregue com sucesso para analisar amostras de ADN a partir de células cultivadas e tecidos de murino. Este artigo de vídeo ajudará outros grupos no estabelecimento de um método eficaz para a detecção e quantificação fiável de 8-oxo-dGuo por CLER-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

O stress oxidativo está associado a diversos processos fisiológicos e patológicos, assim como o metabolismo xenobiótico, levando a oxidação de biomacromoléculas, incluindo ADN. Portanto, a detecção eficiente de oxidação do DNA é importante para uma variedade de disciplinas de investigação, incluindo a medicina e toxicologia. Um biomarcador comum de ADN é danificado oxidativamente 8-oxo-7,8-di-hidro-2&#39;-desoxiguanosina (8-oxo-dGuo; erroneamente frequentemente referido como 8-hidroxi-2&#39;-desoxiguanosina (8-OH-dGuo ou 8 -oxo-dG)). Vários protocolos de medição 8-oxo-dGuo por cromatografia líquida de alta pressão com detecção electroquímica (HPLC-ED) foram descritos. No entanto, estas foram principalmente aplicada a ADN purificado tratados com pró-oxidantes. Além disso, devido a diferenças metodológicas entre laboratórios, principalmente devido às diferenças de equipamento analítico, a adoção de métodos publicados para a detecção de 8-oxo-dGuo por HPLC-ED requer otimização cuidadosa de cada laboratório. UMAprotocolo abrangente, que descreve este processo de otimização, é inexistente. Aqui, um protocolo detalhado é descrito para a detecção de 8-oxo-dGuo por CLER-ED, no DNA a partir de células cultivadas ou tecidos animais. Ele ilustra como a preparação da amostra de ADN podem ser facilmente e rapidamente optimizada para minimizar a oxidação do ADN indesejáveis ​​que podem ocorrer durante a preparação da amostra. Este protocolo demonstra como detectar 8-oxo-dGuo em células humanas cultivadas adenocarcinoma alveolar (isto é, células A549) tratadas com o agente oxidante KBrO 3, e a partir do baço de ratinhos expostos ao hidrocarboneto aromático policíclico dibenzo (DEF, p) criseno (DBC, anteriormente conhecido como dibenzo (a, l) pireno, DalP). Em geral, este trabalho ilustra como uma metodologia de CLER-ED pode ser facilmente optimizado para a detecção de 8-oxo-dGuo em amostras biológicas.

dGuo foi observado a ter um tempo de retenção de 4,7 minutos enquanto que 8-oxo-dGuo teve um tempo de retenção de aproximadamente 6,4 minutos (Figura 2A e B). Há uma diferença de cerca de 1.000 vezes em as alturas dos picos entre os dois analitos, como pode ser visto na Figura 2C. Voltamogramas de 8-oxo-dGuo e dGuo foram obtidos por padrões que funcionam a um potencial de trabalho na gama de 0,2-1,1 V. O potencial de trabalho óptima para o 8-oxo-dGuo foi determi...

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

O objectivo do presente protocolo é a detecção do marcador de oxidação do DNA, 8-oxo-7,8-di-hidro-2&#39;-desoxiguanosina (8-oxo-dGuo) por CLER-ED, no DNA a partir de células cultivadas ou tecidos animais.

A medição por HPLC do ADN Oxidação de biomarcador, 8-oxo-7,8-di-hidro-2&#39;-desoxiguanosina, e em células em cultura de tecidos animais

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

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26.2K visualizações.  Health Canada.  O objectivo do presente protocolo é a detecção do marcador de oxidação do DNA, 8-oxo-7,8-di-hidro-2'-desoxiguanosina (8-oxo-dGuo) por CLER-ED, no DNA a partir de células cultivadas ou tecidos animais. 

Vídeo: HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2’-deoxyguanosine, in Cultured Cells and Animal Tissues

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement 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 of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Steady-state Pré-steady-state, e Single-turnover Medição, Kinetic para DNA glicosilase Atividade

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

Química

Folding and Characterization of a Bio-responsive Robot from 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 stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

Dobragem e Caracterização de um robô Bio-responsivo a partir de ADN de 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

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared spectroscopy is used to predict nondestructively total sugar, total organic acid, and total anthocyanin content in each blueberry. The technique is expected to enable the selection of only delicious blueberries from all harvested ones. The near-infrared absorption spectra of blueberries are measured with the diffuse reflectance mode at the positions not on the calyx. The ingredient contents of a blueberry determined by high-performance liquid chromatography are used to construct models to predict the ingredient contents from observed spectra. Partial least squares regression is used for the construction of the models. It is necessary to properly select the pretreatments for the observed spectra and the wavelength regions of the spectra used for analyses. Validations are necessary for the constructed models to confirm that the ingredient contents are predicted with practical accuracies. Here we present a protocol to construct and validate the models for nondestructive prediction of ingredient contents in blueberries by near-infrared spectroscopy.

We present here a protocol to construct and validate models for nondestructive prediction of total sugar, total organic acid, and total anthocyanin content in individual blueberries by near-infrared spectroscopy.

Construção de Modelos de Previsão Nondestructive de Conteúdos ingrediente em mirtilos por espectroscopia no infravermelho próximo com base em medições de HPLC

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

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

-HPLC baseado Ensaio para monitorar Extracelular Nucleotide / nucleosídeos Metabolismo em crônicas células de leucemia linfocítica Humanos

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

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

Síntese de Bioconjugados de proteínas<em&gt; via</em&gt; Química cisteína-maleimida

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

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, the focus is on metallic substrates immersed in acidic solutions containing organic corrosion inhibitors, as these systems can be particularly sensitive to oxidation following removal from solution. To minimize the likelihood of such degradation, samples are removed from solution within a glove box purged with inert gas, either N2 or Ar. The glove box is directly attached to the load-lock of the ultra-high vacuum X-ray photoelectron spectroscopy instrument, avoiding any exposure to the ambient laboratory atmosphere, and thus reducing the possibility of post immersion substrate oxidation. On this basis, one can be more certain that the X-ray photoelectron spectroscopy features observed are likely to be representative of the in situ submerged scenario, e.g. the oxidation state of the metal is not modified.

A protocol to avoid the oxidation of metallic substrates during sample transfer from an inhibited acidic solution to an X-ray photoelectron spectrometer is presented.

Determinar a composição química do inibidor de corrosão / Metal Interfaces com XPS: Minimizando Pós Immersion Oxidação

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

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

Fabricação e teste dos termómetros fotônicos

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

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

Medição simultânea de HDAC1 e atividade HDAC6 em HeLa células usando 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

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

DNA-magnético partícula análise por dispersão de luz dinâmica e eletroforético de vinculação

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

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

Desembaraçar as fibras de aramida de copolímero de alta resistência para permitir a determinação de suas propriedades mecânicas

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

A medição por HPLC do ADN Oxidação de biomarcador, 8-oxo-7,8-di-hidro-2'-desoxiguanosina, e em células em cultura de tecidos animais

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