Name: evaluating in vitro dna damage using comet assay
Uploaded: 2017-10-11T14:00:00
Description: Watch this Scientific Journal Video about Evaluating In Vitro DNA Damage Using Comet Assay at JoVE.com

Denne forskning blev støttet af murene forskningsprogrammet NIH, national kritisk infrastruktur og CCR. Alle forfattere har modtaget murene forskning tilskud fra NIH, national kritisk infrastruktur og CCR.

1. forberede reagenser <ol><li>1 x PBS <ol><li>fortyndes 100 mL 10 x PBS med 900 mL dH 2 O og ph indstilles til 7.4 ved hjælp af et pH-meter. Opbevares ved stuetemperatur.</li>
	</ol></li> <li>Lysis løsning (LS) <ol><li>forberede 2,5 M NaCl, 100 mM dinatrium EDTA, 10 mM Tris base og 200 mM NaOH i 900 mL dH 2 O, det normalt tager ca 20 min til at Lad blandingen fuldt opløses. PH indstilles til 10 ved hjælp af et pH-meter. Tilføj 1% natrium lauryl sarcosinate ...

<ol>
	<li>Ostling, O., Johanson, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrophoretic+study+of+radiation-induced+DNA+damages+in+individual+mammalian+cells.">Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells.</a> Biochem Biophys Res Commun. 123 (1), 291-298 (1984).</li><li>Singh, N. P., McCoy, M. T., Tice, R. R., Schneider, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+quantitation+of+low+levels+of+DNA+damage+in+individual+cells.">A simple technique for quantitation of low levels of DNA damage in individual cells.</a> Exp Cell Res. 175 (1), 184-191 (1988).</li><li>Tice, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+gel/comet+assay:+guidelines+for+in+vitro+and+in+vivo+genetic+toxicology+testing.">Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.</a> Environ Mol Mutagen. 35 (3), 206-221 (2000).</li><li>Shah, A. J., Lakkad, B. C., Rao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotoxicity+in+lead+treated+human+lymphocytes+evaluated+by+micronucleus+and+comet+assays.">Genotoxicity in lead treated human lymphocytes evaluated by micronucleus and comet assays.</a> Indian J Exp Biol. 54 (8), 502-508 (2016).</li><li>Azqueta, A., Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+comet+assay:+a+comprehensive+guide+to+measuring+DNA+damage+and+repair.">The essential comet assay: a comprehensive guide to measuring DNA damage and repair.</a> Arch Toxicol. 87 (6), 949-968 (2013).</li><li>Goldstein, M., Kastan, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+damage+response:+implications+for+tumor+responses+to+radiation+and+chemotherapy.">The DNA damage response: implications for tumor responses to radiation and chemotherapy.</a> Annu Rev Med. 66, 129-143 (2015).</li><li>Gavande, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+targeted+therapy:+The+past+or+future+of+cancer+treatment?.">DNA repair targeted therapy: The past or future of cancer treatment?.</a> Pharmacol Ther. 160, 65-83 (2016).</li><li>Torgovnick, A., Schumacher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+mechanisms+in+cancer+development+and+therapy.">DNA repair mechanisms in cancer development and therapy.</a> Front Genet. 6, 157 (2015).</li><li>Weston, V. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PARP+inhibitor+olaparib+induces+significant+killing+of+ATM-deficient+lymphoid+tumor+cells+in+vitro+and+in+vivo.">The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo.</a> Blood. 116 (22), 4578-4587 (2010).</li><li>Brown, J. S., O'Carrigan, B., Jackson, S. P., Yap, T. 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Komet assay er et effektivt redskab til at måle enkelt og dobbelt-strenget DNA pauser på cellulært niveau. Analysen har været almindeligt anvendt som en "golden standard" i undersøgelser vedrørende genotoksicitet og bioovervågning13, lige fra base læsioner, DNA krydsbindinger, stof udvikling og alkali følsomme steder. I den foreliggende undersøgelse viste vi to særskilte trinvise protokoller for basisk og neutral komet assays, henholdsvis. Kombinere enkelt celle elektroforese, fluoresce...

Komet analysen blev først udviklet af Ostling og Johanson i 1984 ved at demonstrere migration af DNA fragmenter fra kerner under en neutral tilstand1. Teknikken blev senere udviklet af Singh et al., viser, at en basisk tilstand væsentligt øget specificitet og reproducerbarhed af assay2. Siden da, neutral komet analysen bruges oftest til at opdage dobbelt-strenget DNA pauser, mens basisk komet assay er mere følsomme for mindre mængder af DNA-skader, herunder enkelt og dobbeltstrenget DNA bryder, alkali-labil websteder, DNA-DNA eller DNA-protein cross-linking, og DNA enkelt-strenget pauser tilknyttet inkomplet excision reparation sites3,4. Begge assays give visualisering af fragmenterede DNA og give en enkel måde at vurdere kvantitativt DNA-skader. Komet analysen betragtes som en følsom analysemetode for in vitro og i vivo genetiske toksikologiske undersøgelser og gælder for forskellige forskningsområder, som tidlige lægemiddelkandidat udvalg, miljøovervågning, human bioovervågning, og grundlæggende forskning i DNA-skader og reparation5.
Princippet om analysen er at under et elektrisk felt, fragmenterede DNA vandrer ud af nucleoid organ (også kendt som "komet-hoved") og danner en DNA plet i agarosegel (også kendt som "komet-hale"). Med nukleotid farvning, kan omfanget af DNA-skader kvantificeres ved at analysere "kometer" dannet af denne enkelt celle elektroforese. Beregning af halen øjeblik kan yderligere hjælpe for at sammenligne DNA-skader blandt forskellige eksperimentelle grupper. Sammenlignet med traditionelle metoder til DNA skader påvisning, er comet assay direkte, følsomme, billig og forholdsvis enkel.
Strålebehandling og kemoterapier er fælles strategier til behandling af cancer ved at generere enkelt streng og dobbeltstrenget DNA pauser i kromosomer6. Den seneste avancement i DNA reparation hæmmere giver mulighed for en mere effektiv genotoksisk effekt af kombination kemoterapi, og derfor reducerer potentielt systemiske bivirkninger såsom anæmi, infektion og knoglemarv undertrykkelse7, 8. i denne undersøgelse, vi viste undersøgelsen af en poly (ADP-ribose) polymerase (PARP) hæmmer, olaparib (Ola)9. PARP er en rigelige nukleare protein og er ansvarlig for DNA base excision reparation ved at danne en poly (ADP-ribose) polymer10. Temozolomide (TMZ) er en mundtligt rådighed alkylating agent og har været meget udbredt for gliom patientbehandlingen. Ved hjælp af comet analysen for at kvantificere DNA-skader, vise vi at kombinere olaparib med temozolomide dybt forbedrer DNA-skader i gliom celler, hvilket tyder på olaparib/temozolomide kombinationsterapi er en effektiv strategi til at behandle gliom, sammenlignet med temozolomide alene11.

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			<td height="21" style="height:21px;width:216px;">Reagents</td>
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			<td style="width:167px;"></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:216px;">10x PBS(Ca++, Mg++ free)</td>
			<td style="width:101px;">TEKnova</td>
			<td style="width:119px;">P0196</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">NaCl</td>
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			<td height="21" style="height:21px;width:216px;">EDTA</td>
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			<td height="21" style="height:21px;width:216px;">Trizma base</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Low melting point agarose</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
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			<td height="42" style="height:42px;width:216px;">Horizontal electrophoresis chamber</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescent microscope</td>
			<td style="width:101px;">Zeiss</td>
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			<td height="21" style="height:21px;width:216px;">Micropipettor</td>
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	</tbody>
</table>

evaluating in vitro dna damage using comet assay

DNA-skader er et almindeligt fænomen for hver celle i løbet af sin levetid, og er defineret som en ændring af den kemiske struktur af genomisk DNA. Kræft behandlinger, såsom radio- og kemoterapi, indføre enorme mængde ekstra DNA-skader, fører til celle cyklus anholdelse og apoptose at begrænse kræft progression. Kvantitativ vurdering af DNA-skader i eksperimentel kræftbehandling er et vigtigt skridt til at retfærdiggøre effektiviteten af en genotoksisk agent. I denne undersøgelse, vi fokuserer på en enkelt celle elektroforese assay, også kendt som komet-analysen, der kan kvantificere enkelt og dobbelt-strenget DNA pauser i vitro. Komet assay er et DNA skader kvantificering metode, som er effektiv og nem at udføre, og har lav tid/budget krav og høj reproducerbarhed. Her, fremhæve vi nytten af comet assay prækliniske undersøgelser ved at evaluere genotoksisk virkning af olaparib/temozolomide kombinationsbehandling til U251 gliom celler.

Denne protokol beskriver en trinvis arbejdsproces for comet assay udførelse og analyse af data (figur 1). Resultater fra alkaline og neutral komet assays viste, at kometen halen af doxorubicin-behandlede U251 celler (1 µM, 20 h) blev længere og havde højere DNA intensitet, hvilket tyder på en betydelig ophobning af fragmenterede DNA som følge af kemoterapi (figur 2).
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Watch this Scientific Journal Video about Evaluating In Vitro DNA Damage Using Comet Assay at JoVE.com

Evaluating In Vitro DNA Damage Using Comet Assay

Evaluering af In Vitro DNA skader med Comet Assay

Komet assay er en effektiv metode til at påvise DNA skade herunder enkelt og dobbelt-strenget DNA pauser. Vi beskriver alkalisk og neutral komet assays for at måle DNA skader i kræftceller til at evaluere den terapeutiske effekt af kemoterapi.

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

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