מחקר זה מומן על ידי משרד הבריאות הקנדית ג&#39;נומיקס המחקר ופיתוח היוזמה (GRDI) ותקינת האסטרטגיה הקנדית לביוטכנולוגיה (CRSB). יש הסופרים אין ניגוד האינטרסים.

<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.

למרות 8-אוקסו-dGuo דווח כסמן ביולוגי שימושי של חמצון ה- DNA, הכימות אמין שלה יכול להוות אתגר. למרות כמה שיטות שפורסמו קיימות, יש צורך בסקירה מקיפה, תיאורים של פרוטוקול להתיר לחוקרים לפרוס את השיטה במעבדות שלהם. כאן אנו מציגים סקירה מפורטת של פרוטוקול מבוסס HPLC שיאפשר למשתמש...

מינים תגובתי חמצן (ROS), מצב יציב שיכולים להגדיל את הרמות בהרבה מצבים פתולוגיים וחילוף חומרי xenotoxic, תורמים לשכיחות מוגברת של נזק לדנ&quot;א חמצוני. בין כמה מוצרי חמצון nucleobases האפשרי, נזק לדנ&quot;א חמצוני יכול בקלות למדוד באמצעות הסמן היציב 8-אוקסו-7,8-Dihydro-2&#39;-deoxyguanosine (8-אוקסו-dGuo), שהוא אחת מצורות חמצון של 2 &#39; -deoxyguanosine (dGuo) 1. 8-אוקסו-dGuo הוא נגע DNA הנפוץ ביותר 2, ולכן, כבר למד לפירוט רב יותר כסמן ביולוגי חמצון ה- DNA למרות קיומם של מוצרי חמצון ה- DNA המרובים 3. בבני אדם, נזק זה ניתן לתיקון באמצעות תיקון כריתת בסיס על ידי glycosylase 8-oxoguanine 1 (hOGG1) 4. אם עזב מתוקן, 8-אוקסו-dGuo יכול לתרום להיווצרותן של מוטציות זוג-החלפת בסיס (כלומר, G לT transversions) 4. חשוב לציין, 8-אוקסו-dGuo הוא סמן הוקם FOנזק לדנ&quot;א r ביחס לייזום והקידום של היווצרות הסרטן 2. לכן, כימות מדויק של 8-אוקסו-dGuo הוא סמן ביולוגי שימושי ורצוי של נזק לדנ&quot;א חמצוני 5. יש בלבול נפוץ בספרות בנוגע לשמות הנכונים לצורות oxidatively פגועות של 2-deoxyguanosine ו, יתר על כן, את השם הנכון של המתחם (ים) שנמדדו באופן שגרתי כסמן ביולוגי של נזק לדנ&quot;א חמצוני 6. צורות tautomeric 8-קטו 6,8-diketo ו6-enol, של 8-אוקסו-dGuo (מוצג באיור 1) הן שני tautomers הבולט ביותר שנדון בספרות 5,7. טופס 6,8-diketo הוא הצורה הבולטת ביותר ב- pH הפיזיולוגי של 7.4, והוא מוצר חמצון ה- DNA הבולט 7. לכן, 8-אוקסו-dGuo, במקום 8-הידרוקסי-dGuo הוא השם המתאים ביותר למוצר חמצון זה 6. כמו כן, חשוב לשים לב ש2-deoxyguanosine (dGuo), ולא nucleobגואנין ASE (גואה) או guanosine ribonucleoside (גואו), בהתאמה, הוא זוהה על ידי רוב השיטות 6. איתור וכימות של 8-אוקסו-dGuo מדויקים הוא מאתגר עקב: השתנות i) בעיכול של דגימת DNA, ii) חמצון adventitious של dGuo 8-אוקסו-dGuo שיכול להתרחש במהלך הכנת מדגם, וiii) את הצורך לאימות יעילה של שיטת HPLC-ED אנליטיים 8. בפרוטוקול זה, אנו חותרים להשגת i) על ידי מתן תנאים, נוחים לעיכול DNA מלא וii) על ידי chelator הכללת המתכת ופתרונות שטופל chelator ומגיב בידוד-דנ&quot;א מיוחד, תוך iii) היה ממוען רק באופן חלקי על ידי הכללת בקרות חיוביות וכך לספק כי השיטה היא מסוגלת לאתר 8-אוקסו-dGuo בדגימות ביולוגיות. אימות נוספת היא מעבר להיקף של מאמר זה. עם זאת, אנו בטוחים כי פרוטוקול זה יעזור לי פוטנציאלימשתמשים לקבוע את המידה שבה הם צריכים לאמת באופן רשמי בפרוטוקול, בהתאם לצרכיהם. רשימה של צעדים הנדרשים לאימות הפורמאלית של השיטה מסופקת נוספת. במהלך הפיתוח והפריסה של שיטה לזיהוי 8-אוקסו-dGuo, שיטות שפורסמו נסקרו ומאוחדת. לכן, שיטה זו מבטלת את הצורך לאסוף מידע ממספר מקורות שפורסמו, כי לעתים קרובות חסרי פרטים ניסיוניים חשובים גם בעת מתן אמצעי מהיר ופשוט של בדיקות אם השיטה לאיתור והכימות של 8-אוקסו-dGuo כבר אימצה בהצלחה. שיטה מותאמת זה הועסקה לנתח בהצלחה דגימות DNA מהתאים בתרבית רקמה ועכברית. מאמר זה וידאו יסייע קבוצות אחרות בהקמת שיטה יעילה לגילוי אמין וכימות של 8-אוקסו-dGuo ידי HPLC-ED.

<table><tbody><tr><td>8-oxo-dGuo standard</td><td>Cayman Chemical Company</td><td>89320</td><td>Inappropriately referred to as &quot;8-hydroxy-2&#039;-deoxy Guanosine&quot;; see &lt;strong&gt;Fig. 1&lt;/strong&gt; and text for details.</td></tr><tr><td>Alkaline phosphatase</td><td>Sigma-Aldrich</td><td>P5931</td><td>From &lt;em&gt;E. coli&lt;/em&gt;</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 (&lt;em&gt;e.g.&lt;/em&gt; with 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&amp;nbsp;</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

סטרס חמצונים קשור הרבה תהליכים פיסיולוגיים ופתולוגיים, כמו גם חילוף חומרים xenobiotic, מוביל לחמצון של Biomacromolecules, כולל DNA. לכן, זיהוי יעיל של חמצון ה- DNA הוא חשוב עבור מגוון רחב של תחומי מחקר, כוללים רפואה וטוקסיקולוגיה. סמן ביולוגי משותף של דנ&quot;א פגום oxidatively הוא 8-אוקסו-7,8-Dihydro-2&#39;-deoxyguanosine (8-אוקסו-dGuo; לעתים קרובות מכונה בטעות כ8-הידרוקסי-2&#39;deoxyguanosine (8-OH-dGuo או 8 -oxo-DG)). כמה פרוטוקולים ל8-אוקסו-dGuo מדידה על ידי כרומטוגרפיה נוזלית בלחץ גבוה עם זיהוי אלקטרוכימי (HPLC-ED) תוארו. עם זאת, אלה יושמו בעיקר ל- DNA מטוהר שטופל בפרו-חמצון. בנוסף, עקב הבדלים מתודולוגיים בין מעבדות, בעיקר בשל הבדלים בציוד אנליטי, האימוץ של שיטות שפורסמו לצורך זיהוי של 8-אוקסו-dGuo ידי HPLC-ED דורש אופטימיזציה זהירה בכל מעבדה.פרוטוקול מקיף, המתאר תהליך האופטימיזציה כזה, חסר. כאן, פרוטוקול מפורט מתואר לגילוי של 8-אוקסו-dGuo ידי HPLC-ED, בDNA מהתאים בתרבית או רקמות של בעלי חיים. זה מדגים כיצד הכנת דגימת DNA יכולה להיות מותאמת בקלות ובמהירות כדי למזער חמצון ה- DNA לא רצוי שיכול להתרחש במהלך הכנת מדגם. פרוטוקול זה מראה כיצד לזהות 8-אוקסו-dGuo בתאים בתרבית אדם אדנוקרצינומה מכתשי (כלומר, תאי A549) שטופלו בסוכן חמצון KBrO 3, ומהטחול של עכברים שנחשפו לdibenzo פחמימנים ארומטיים polycyclic (def, עמ &#39;) chrysene (DBC, שנקרא בעבר פיר dibenzo (, יב), DalP). בסך הכל, עבודה זו מדגימה כיצד מתודולוגיה HPLC-ED יכולה להיות מותאמת בקלות לזיהוי של 8-אוקסו-dGuo בדגימות ביולוגיות.

dGuo נצפה ליש זמן שמירה של 4.7 דקות ואילו 8-אוקסו-dGuo זמן שמירה של כ 6.4 דקות (איור 2 א &#39;וב&#39;). יש הבדל כ -1,000 פי בגבה השיא בין שני analytes, כפי שניתן לראות באיור 2 ג. Voltammograms ל8-אוקסו-dGuo וdGuo התקבלו על ידי סטנדרטים פועלים בפוטנציאל עבודה בטווח של 0.2-1.1 V. פוטנציאל העב?...

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-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

המטרה של פרוטוקול זה היא הגילוי של סמן חמצון ה- DNA, 8-אוקסו-7,8-Dihydro-2&#39;-deoxyguanosine (8-אוקסו-dGuo) על ידי HPLC-ED, בDNA מהתאים בתרבית או רקמות של בעלי חיים.

HPLC המדידה של ה- DNA חמצון ביומרקר, 8-אוקסו-7,8-Dihydro-2&#39;-deoxyguanosine, בתאים בתרבית ורקמות בעלי החיים

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

hplc-measurement-dna-oxidation-biomarker-8-oxo-78-dihydro-2

Research

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Chemistry

26.3K Views.  Health Canada.  המטרה של פרוטוקול זה היא הגילוי של סמן חמצון ה- DNA, 8-אוקסו-7,8-Dihydro-2'-deoxyguanosine (8-אוקסו-dGuo) על ידי HPLC-ED, בDNA מהתאים בתרבית או רקמות של בעלי חיים. 

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

Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.

Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.

רדיקלים חופשיים בביולוגיה כימית: מהתנהגות כימית לפיתוח סמנים ביולוגיים

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological ...

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.

מדידת מצב יציב, טרום מצב יציב, וחד מחזור קינטי לפעילות ה-DNA Glycosylase

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

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).

מתקפל ואפיון של רובוט ביו-תגובה מה- DNA אוריגמי

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

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 מבוססי Assay לפקח תאיים נוקלאוטיד / מטבוליזם nucleoside בתאי לוקמיה לימפוציטית כרונית

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.

סינתזה של חלבון Bioconjugates<em&gt; דרך</em&gt; כימיה ציסטאין-maleimide

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.

קביעת ההרכב הכימי של ממשקי קורוזיה מונעים / מתכת עם XPS: חמצון טבילת הודעת מזעור

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.

ייצור ובדיקה של מדי חום פוטוני

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.

המדידה בו זמנית HDAC1 ופעילות HDAC6 הלה תאים באמצעות 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 ...

קביעת מידת הנזק החמצוני לדנ"א בתאי MCF-7

Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells

8-Oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly impacts gene expression and serves as a pivotal factor in stimulating neurodegenerative disorders, cancer, and aging. Therefore, precise quantification of 8-oxoG has clinical significance in the investigation of DNA damage detection methodologies. However, at present, the existing approaches for 8-oxoG detection pose challenges in terms of convenience, expediency, affordability, and heightened sensitivity. We employed the sandwich enzyme-linked immunosorbent assay (ELISA) technique, a highly efficient and swift colorimetric method, to detect variations in 8-oxo-dG content in MCF-7 cell samples stimulated with different concentrations of hydrogen peroxide (H2O2). We determined the concentration of H2O2 that induced oxidative damage in MCF-7 cells by detecting its IC50 value in MCF-7 cells. Subsequently, we treated MCF-7 cells with 0, 0.25, and 0.75 mM H2O2 for 12 h and extracted 8-oxo-dG from the cells. Finally, the samples were subjected to ELISA. Following a series of steps, including plate spreading, washing, incubation, color development, termination of the reaction, and data collection, we successfully detected changes in the 8-oxo-dG content in MCF-7 cells induced by H2O2. Through such endeavors, we aim to establish a method to evaluate the degree of DNA oxidative damage within cell samples and, in doing so, advance the development of more expedient and convenient approaches for DNA damage detection. This endeavor is poised to make a meaningful contribution to the exploration of associative analyses between DNA oxidative damage and various domains, including clinical research on diseases and the detection of toxic substances.

זרקור המחבר: הערכה כמותית של 8-oxo-dG בתאי MCF-7 באמצעות ELISA

This protocol describes an efficient method for quantitatively detecting DNA oxidative damage in MCF-7 cells by ELISA technology.

כימות הרמה של 8-oxo-dG באמצעות בדיקת ELISA להערכת נזק חמצוני לדנ"א בתאי MCF-7

8-Oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly ...

HPLC המדידה של ה- DNA חמצון ביומרקר, 8-אוקסו-7,8-Dihydro-2'-deoxyguanosine, בתאים בתרבית ורקמות בעלי החיים

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Chapters in this video

רדיקלים חופשיים בביולוגיה כימית: מהתנהגות כימית לפיתוח סמנים ביולוגיים

מדידת מצב יציב, טרום מצב יציב, וחד מחזור קינטי לפעילות ה-DNA Glycosylase

מתקפל ואפיון של רובוט ביו-תגובה מה- DNA אוריגמי

HPLC מבוססי Assay לפקח תאיים נוקלאוטיד / מטבוליזם nucleoside בתאי לוקמיה לימפוציטית כרונית

סינתזה של חלבון Bioconjugates

קביעת ההרכב הכימי של ממשקי קורוזיה מונעים / מתכת עם XPS: חמצון טבילת הודעת מזעור

ייצור ובדיקה של מדי חום פוטוני

המדידה בו זמנית HDAC1 ופעילות HDAC6 הלה תאים באמצעות UHPLC-MS

כימות הרמה של 8-oxo-dG באמצעות בדיקת ELISA להערכת נזק חמצוני לדנ"א בתאי MCF-7

כימות הרמה של 8-oxo-dG באמצעות בדיקת ELISA להערכת נזק חמצוני לדנ"א בתאי MCF-7