This research was supported by the R&#38;D Program for the Society&#160;of the National Research Foundation (NRF), funded by the Ministry of Science, ICT &#38; Future Planning (Grant No. 2013M3C8A1075908).

1. DNA Extraction from FFPE Reference Standard Cell Lines
Note: For this procedure, DNA extraction was performed from FFPE reference standard cell lines (HD598, HD593, HD617, HD273 and wildtype (WT)) using the FFPE Tissue DNA isolation kit as described in the protocol below. Automated DNA extraction was achieved by following the manufacturer&#39;s instructions for total DNA isolation.
<ol>
	<li>Using a microtome and the original FFPE block, manually prepare fresh sections prior to DNA extrac...

<ol>
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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=BRAF+and+RAS+mutations+in+human+lung+cancer+and+melanoma.">BRAF and RAS mutations in human lung cancer and melanoma.</a> Cancer research. 62, 6997-7000 (2002).</li><li>Wang, L., 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=BRAF+mutations+in+colon+cancer+are+not+likely+attributable+to+defective+DNA+mismatch+repair.">BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair.</a> Cancer research. 63, 5209-5212 (2003).</li><li>Hindson, B. 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C., 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=Highly+precise+measurement+of+HIV+DNA+by+droplet+digital+PCR.">Highly precise measurement of HIV DNA by droplet digital PCR.</a> PloS one. 8, e55943 (2013).</li><li>Miotke, L., Lau, B. T., Rumma, R. T., Ji, H. 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Myung Ryuri Oh, Si Eun Kim, and Young Deug Kim are employees of ABION CRO.

Here, we highlight the applicability of ddPCR and DNA isolation from FFPE reference standard cell line samples for a specific gene mutation assessment. In this study, TPS automated DNA isolation method is used which can be readily adapted, automated, and can accommodate up to 48 different samples simultaneously, allowing for larger scale experiments and lower variability. One of the limitations of the DNA isolation in the present work is that every FFPE sample is unique, and will vary one another in surface contaminants,...

The accumulation of genetic mutations in key regulatory genes alters normal cell programing like cell proliferation, differentiation, and survival, leading to cancer1. The RAS-RAF-MAP kinase pathway mediates cellular responses to growth signals. Oncogenic BRAF mutations can result from driver mutations in the&#160;BRAF&#160;gene, which may cause the BRAF oncoprotein to become overactive2.&#160;Mutations in the&#160;BRAF&#160;gene also result in overactive downstream signaling via MEK and ERK3, which, in turn, leads to excessive cell growth and proliferation independently of growth&#160;factor-mediated regulation4-6.
Several tools are available for DNA mutation profiling, such as quantitative real-time&#160;BRAF V600E mutations in formalin-fixed, paraffin-embedded (FFPE) reference standard cell lines by ddPCR. ddPCR is an PCR-based method for absolute quantification offering higher accuracy compared to conventional quantitative real-time PCR (qPCR)7,8. ddPCR also provides higher resolving power and accuracy for the detection of rare mutations in DNA templates, enabling more informative cancer research and diagnosis9. Additional advantages of ddPCR over conventional qPCR include its enhanced sensitivity and accuracy when studying low template copy numbers10-12. Herein, a protocol for automatically extracting DNA from FFPE reference standard cell lines, followed by determining the presence or absence of BRAF V600E mutations by ddPCR is demonstrated. The usage of software for data analysis and a graphical representation of the results are also described. The entire procedure is relatively simple and totally depends on the number of samples to be profiled and the number of conventional PCR and ddPCR machines available.
The following protocol describes standard procedures for BRAF V600E-positive FFPE reference standard cell lines (HD598, HD593, HD617, HD273 and wildtype (WT)) is performed in a fully automated instrument using the Tissue Preparation System (TPS) protocol. Subsequently, isolated DNA samples are analyzed for the presence of BRAF V600E mutations using ddPCR system. Targeted mutation analysis is performed after all samples have been profiled and the data has been loaded into the data analysis software. Depending on the number of samples/groups studied, data analysis may require from one to several hours. The experimental component of the methodology requires accuracy in handling&#160;<a href="http://www.jove.com/science-education/5096/isolating-nucleic-acids-from-yeast" title="Isolating Nucleic Acids from Yeast, a JoVE Science Education video explaining more about about the context of RNA">DNA</a>&#160;and <a href="http://www.jove.com/science-education/5034/introduction-to-serological-pipettes-and-pipettors" title="Introduction to Serological Pipettes and Pipettors, a JoVE Science Education video explaining more about about the context of pipetting">pipetting</a>&#160;into 96 well plates, while data analysis is performed using 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.

For our ddPCR analysis, we studied the BRAF V600E mutation FFPE reference standard cell lines. The droplet reader connects to a laptop computer running data analysis software. Each individual droplet is defined on the basis of fluorescent amplitude as being either positive or negative. The software provided by manufacturer also allows a user-defined cutoff to be entered to define the threshold between the positive and negative droplets. The number of positive and negative droplets in a sample is used to calculat...

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

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

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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Research

JoVE Journal

Biology

13.1K visualizações.  Seoul National University.  O objetivo deste vídeo é para demonstrar como executar extração de DNA automatizado a partir de linhas fixas em formalina e embebidos em parafina (FFPE) de referência padrão de células e de gotículas digital de análise de PCR (ddPCR) para detectar mutações raras em um ambiente clínico. Detecção de mutações em amostras de FFPE demonstra a utilidade clínica de ddPCR em amostras FFPE. 