Name: employing digital droplet pcr to detect braf v600e mutations in formalin-fixed paraffin-embedded reference standard cell lines
Uploaded: 2015-10-08T14:00:00
Description: Watch this Scientific Journal Video about Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines at JoVE.com

Denne forskning blev støttet af F &amp; U-program for Society of National Research Foundation (NRF), finansieret af Ministeriet for Videnskab, IKT &amp; fremtidige planlægning (Grant nr 2013M3C8A1075908).

1. DNA ekstraktion fra FFPE Reference Standard cellelinier Bemærk: Til denne procedure, blev DNA-ekstraktion udføres fra FFPE referencestandard cellelinier (HD598, HD593, HD617, HD273 og vildtype (WT)) ved hjælp af FFPE Tissue DNA isolation kit, som beskrevet i protokollen nedenfor. Automatiseret DNA ekstraktion blev opnået ved at følge producentens instruktioner for total DNA-isolering. <ol><li> Ved hjælp af en mikrotom og den oprindelige FFPE blokken, manuelt forberede friske sekti...

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Her fremhæver vi anvendeligheden af ​​ddPCR og DNA isolation fra FFPE referencestandard celle line prøver for en bestemt genmutation vurdering. I denne undersøgelse, er TPS automatiseret DNA-isolering anvendte metode, som let kan tilpasses, automatiseret og kan rumme op til 48 forskellige prøver samtidigt, hvilket muliggør større skala eksperimenter og lavere variabilitet. En af begrænsningerne af DNA-isolering i det foreliggende arbejde er, at hver FFPE prøven er unikt og vil variere hinanden i overfladefor...

Ophobningen af genetiske mutationer i vigtige regulatoriske gener ændrer normal celle programmering ligesom celledeling, differentiering og overlevelse, hvilket fører til kræft 1. RAS-RAF-MAP-kinase pathway medierer cellulære reaktioner på vækstsignaler. Onkogen BRAF mutationer kan skyldes førerens mutationer i BRAF-genet, som kan forårsage BRAF oncoprotein at blive overaktiv 2. Mutationer i BRAF-genet også resultere i overaktiv nedstrøms signalering via MEK og ERK 3, som til gengæld fører til overdreven vækst og proliferation uafhængigt af vækstfaktor-medieret regulering 4-6. Flere værktøjer til rådighed for DNA-mutation profilering, såsom kvantitative realtid BRAF V600E mutationer i formalin-fikseret, paraffinindlejrede (FFPE) referencestandard cellelinjer ved ddPCR. ddPCR er en PCR-metode til absolut kvantificeringtilbyde højere nøjagtighed i forhold til konventionelle kvantitativ real-time PCR (qPCR) 7,8. ddPCR giver også højere opløsningsevne og nøjagtighed til påvisning af sjældne mutationer i DNA-skabeloner, der muliggør mere informativ kræftforskning og diagnose 9. Yderligere fordele ved ddPCR i forhold til konventionelle qPCR omfatter dens forbedret følsomhed og nøjagtighed, når studerer lav skabelon kopiantal 10-12. Heri er en protokol til automatisk ekstraktion af DNA fra FFPE referencestandard cellelinjer, efterfulgt af bestemmelse af tilstedeværelsen eller fraværet af BRAF V600E mutationer ved ddPCR påvist. Brugen af ​​software til dataanalyse og en grafisk gengivelse af resultaterne er også beskrevet. Hele proceduren er forholdsvis enkel og fuldstændig afhænger af antallet af prøver, der skal profilerede og antallet af konventionel PCR og ddPCR maskiner til rådighed. Følgende protokol beskriver standardprocedurer for BR<html> AF V600E-positive FFPE referencestandard cellelinjer (HD598, HD593, HD617, HD273 og vildtype (WT)) udføres i en fuldautomatisk instrument ved hjælp af Preparation System (TPS) protokol Tissue. Efterfølgende isolerede DNA-prøver analyseret for tilstedeværelsen af BRAF V600E mutationer ved hjælp ddPCR system. Målrettet mutation analyse udføres efter alle prøver er blevet profileret, og data er blevet indlæst i dataanalyse software. Afhængig af antallet af prøver / undersøgte grupper, dataanalyse kan kræve fra en til flere timer. Den eksperimentelle del af metoden kræver nøjagtighed i håndtering <a href="http://www.jove.com/science-education/5096/isolating-nucleic-acids-from-yeast" title="Isolering nukleinsyrer fra gær, en JOVE Science Education video, der forklarer mere om om forbindelse af RNA">DNA</a> ogrologimaster Pipetter og pipetterne, en JOVE Science Education video, der forklarer mere om om sammenhæng med pipettering &quot;&gt; pipettering plader med 96 brønde, mens data analyse udføres ved hjælp af software.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>&#160;Hamilton MICROLAB STARlet IVD instrument</td>
			<td>Siemens</td>
			<td>10701001</td>
			<td>Automated DNA isolation instrument</td>
		</tr>
		<tr>
			<td>QX200 Droplet Generator&#160;</td>
			<td>Bio-Rad</td>
			<td>772BR1119</td>
			<td></td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader</td>
			<td>Bio-Rad</td>
			<td>771BR1497</td>
			<td></td>
		</tr>
		<tr>
			<td>Conventional PCR machine capable of ramp-time adjustment</td>
			<td></td>
			<td>621BR17718</td>
			<td></td>
		</tr>
		<tr>
			<td>PX1 PCR plate sealer</td>
			<td>Bio-Rad</td>
			<td>770BR1575</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantaSoft software</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA isolation kit&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VERSANT Tissue Preparation Reagents Box 1&#160;</td>
			<td>Siemens</td>
			<td>10632398</td>
			<td></td>
		</tr>
		<tr>
			<td>VERSANT Tissue Preparation Reagents Box 1&#160;</td>
			<td>Siemens</td>
			<td>10632399</td>
			<td></td>
		</tr>
		<tr>
			<td>CO-RE tips</td>
			<td>Siemens</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR mutation analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Supermix&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>BR186-3010</td>
			<td>2X concentration</td>
		</tr>
		<tr>
			<td>DG8 cartridge&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>BR186-4008</td>
			<td></td>
		</tr>
		<tr>
			<td>Droplet Generator oil</td>
			<td>Bio-Rad&#160;</td>
			<td>BR-186-3005</td>
			<td></td>
		</tr>
		<tr>
			<td>Gasket</td>
			<td>Bio-Rad&#160;</td>
			<td>BR186-3006</td>
			<td></td>
		</tr>
		<tr>
			<td>Droplet reader oil</td>
			<td>Bio-Rad&#160;</td>
			<td>BR-186-3004</td>
			<td></td>
		</tr>
	</tbody>
</table>

employing digital droplet pcr to detect braf v600e mutations in formalin-fixed paraffin-embedded reference standard cell lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

Til vores ddPCR analyse, vi studerede BRAF V600E mutation FFPE referencestandard cellelinjer. Dråben læser forbinder til en bærbar computer, der kører dataanalyse software. Hver enkelt dråbe defineres på grundlag af fluorescerende amplitude som værende enten positiv eller negativ. De oplysninger, som producenten software giver også mulighed en brugerdefineret cutoff skal indtastes for at definere grænsen mellem de positive og negative dråber. Antallet af positive og negative dråber i en prøve anvend...

Watch this Scientific Journal Video about Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines at JoVE.com

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

Målet med denne video er at vise, hvordan du udfører automatiseret DNA-ekstraktion fra formalin-fikseret paraffinindlejrede (FFPE) referencestandard cellelinjer og digital dråbe PCR (ddPCR) analyse til påvisning af sjældne mutationer i et klinisk miljø. Detektion af mutationer i FFPE prøver viser den kliniske anvendelighed af ddPCR i FFPE prøver.

Anvender digitale Droplet PCR til Detect BRAF V600E Mutationer i formalinfikserede paraffinindlejrede Reference Standard cellelinier

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

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