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 extraction and analysis, according to established procedures. Ensure that the sample input for the tissue section(s) analyzed does not exceed a combined total thickness of 10 &#181;m and that the surface area does not exceed 600 mm2 for a single section.</li>
</ol>
1.2 TPS protocol
Note: The volumes shown in Table 1 correspond to the minimum required to process 48 samples, and the procedure shown is in accordance with the TPS guidelines. Before starting the experiment settle down the FFPE samples in the e-tube by centrifugation&#160;at 600 x g, to avoid loss of samples during the automated program.
<ol>
	<li>Turn on the automated DNA isolation instrument and computer. Open the Run control software and insert an auto load tray into the TPS deck loading area.</li>
	<li>Dispense reagents into their corresponding troughs as shown in Figure&#160;1.
	<ol>
		<li>Place the 4 samples (BRAF WT, BRAF V600E 50%, 5% and 1%) in the sample carrier racks.</li>
		<li>Place the tip boxes in the troughs in columns 2 and 3 and check the tips for presence of more than one filter per tip, which will abort the run.</li>
		<li>Ensure proper mixing of the lysis buffer and wash buffer by inverting them 3&#8211;5 times and load them into the respective troughs in column 4 ( Figure&#160;1).</li>
		<li>After inverting for few times or mild vortexing, load the elution buffer, magnetic beads, and FFPE buffer in the small troughs in column 5, leaving 1 slot empty where indicated ( Figure&#160;1, Table&#160;1).</li>
		<li>Load a 2&#160;ml&#160;deep well plate (DWP) onto the plate carrier (Figure&#160;1).</li>
	</ol>
	</li>
	<li>Perform the following final steps before starting a run:
	<ol>
		<li>Uncap all tubes and reagent troughs. Confirm that sufficient capacity is available in the liquid waste bottle. Confirm that the solid waste bottle is empty and lined with a biohazard bag. Confirm that the tip eject plate is centered in the waste assembly.</li>
		<li>Close the front cover.</li>
	</ol>
	</li>
	<li>Start the software. Open the NA_Prep_Main_MLSTARlet.med file.</li>
	<li>Click &#8220;Start.&#8221; The instrument status will switch from idle to running.</li>
	<li>Enter the number of samples for this run. Choose the desired method for this run (DNA = 0). Enter the position of the first high volume tip, selecting &#8220;1&#8221; if all trays are full. Enter the position of the first standard volume tip, selecting &#8220;1&#8221; if all trays are full. 
	Note: The instrument will then run through automated steps without user intervention. A detailed workflow is shown in Figure&#160;2. Once the experiment is finished, reagent waste and tips are injected into the waste assembly.</li>
	<li>Quantify purified genomic DNA by using a fluorometric method. 
	Note: DNA samples which contain a minimum concentration of 3.3 ng/&#181;l&#160;are subjected to ddPCR analysis (section 2).</li>
</ol>
2. DNA Mutation Profiling: ddPCR Protocol
Note: The protocol for DNA mutation profiling consists of 3 major steps:1) Droplet generation, 2) Conventional PCR amplification, 3) Droplet reading and 4) DNA mutation profiling.
2.1. Droplet generation
Note: ddPCR supermix is recommended for ddPCR, as this mix contains reagents required for droplet generation.
<ol>
	<li>To avoid contamination, follow standard precautions, such as wearing gloves, using a clean PCR hood, clean pipets, and low-protein-binding tubes.</li>
	<li>Ensure that the minimum concentration of human genomic DNA sample is 3.3 ng/&#181;l. Note: The amount of purified DNA sample concentration will vary based on the analysis of % mutation frequency detection.</li>
	<li>Assemble reaction mixtures in 96-well PCR plates. Thaw and equilibrate the reaction components to RT. Prepare PCR reactions by combining 2X ddPCR supermix (10 &#181;l) and 20 primers (forward and reverse, 900 nM) and probe (250 nM) with each purified DNA sample (66ng/2 &#181;l) make up to 20 &#181;l&#160;with distilled water (as per the droplet generator protocol)</li>
	<li>Vortex the mix thoroughly to ensure homogeneity and briefly centrifuge to collect the contents at the bottom of the tube before dispensing. Load 20 &#181;l&#160;onto the cartridge for droplet formation.</li>
	<li>Operation of the droplet generator, per the manufacturer&#8217;s recommended protocol
	<ol>
		<li>Insert the cartridge into the holder with the notch in the cartridge positioned on the upper left side of the holder. Add 20 &#181;l&#160;of reaction mix containing samples into the middle, and 70 &#181;l&#160;of generator oil into the bottom wells.</li>
		<li>Attach the gasket across the top of the cartridge. Ensure that the gasket is securely hooked on both ends of the holder; otherwise, sufficient pressure for droplet generation will not be achieved.</li>
		<li>Open the droplet generator by pressing the green button on the top of the instrument and insert the cartridge. When the holder is in the correct position, both the power (left right) and holder (middle right) indicator lights are green.</li>
		<li>Press the top button on the instrument again to close the door and initiate droplet generation. Note: After pressing the button, a manifold positions itself over the outlet wells, drawing oil and samples through the microfluidic channels, where droplets are created. Droplets flow into the droplet well, where they accumulate. The droplet indicator light (at right) flashes green after 10 sec to indicate that droplet generation is in progress.</li>
		<li>When droplet generation is complete, all 3 indicator lights change to solid green; open the door by pressing the button, and remove the holder from the unit. Remove the disposable gasket from the holder and discard it. Note: The top wells of the cartridge contain droplets, and the middle and lower wells are nearly empty, with a small amount of residual oil.</li>
	</ol>
	</li>
</ol>
2.2. Preparation for PCR
<ol>
	<li>For each sample, pipet out 40 &#181;l&#160;of the droplet contents from the top well the cartridges into a single well of a recommended 96-well PCR plate as indicated in the manufacturer&#8217;s instrument protocol. Note: Using a multi-channel pipette is ideal for transferring the droplets emulsions. Slow and gentle aspiration of droplets is recommended to minimize droplet shearing during transfers.</li>
	<li>Seal the PCR plate with foil immediately after transferring droplets to avoid evaporation. Use pierceable foil plate seals that are compatible with the PCR plate sealer and the needles in the droplet reader. Follow the instructions in the PCR plate sealer instruction manual.</li>
	<li>Set the plate sealer temperature to 180 &#176;C and the time to 5 sec.</li>
	<li>Touch the arrow to open the tray door. Position the support block on the tray with the 96-well side facing up. Place the 96-well plate onto the support block and ensure that all plate wells are aligned with the support block.</li>
	<li>Cover the 96-well plate with 1 sheet of pierceable foil seal. Once the 96-well plate is secured on the support block and covered with the pierceable foil seal, touch the seal button. The tray will close and heat sealing will initiate.</li>
	<li>When heat sealing is complete, the door opens automatically; remove the plate from the heat block for thermal cycling and then remove the heat block.</li>
	<li>Ensure that all the wells in the plate are sealed by checking that depressions in the foil are readily apparent over each well. Once sealed, the plate is ready for thermal cycling.</li>
	<li>Once droplets are removed, press the latches on the cartridge holder to open it. Remove the empty cartridge and discard it.</li>
	<li>Perform conventional PCR amplification by following the parameters detailed in&#160; Table 2.</li>
</ol>
2.3 Droplet reading (as per the manufacturer&#8217;s recommended protocol)
Note: Following PCR amplification of the nucleic acid target in the droplets, the droplet reader instrument analyzes each droplet individually using a 2-color detection system13. We typically set to detect FAM and VIC reporter fluorophores.
<ol>
	<li>Click &#8220;Flush system&#8221; to prime the droplet reader and make it ready for ddPCR analysis.</li>
	<li>Load the plate into the droplet reader and click &#8220;start.&#8221; The droplet reader aspirates each sample, singulates the droplets, and streams them in single file past a 2-color detector. The detector reads the droplets to enumerate positive and negative samples.</li>
	<li>When droplet reading is complete, open the door and remove the plate holder from the unit. Remove the 96-well PCR plate from the holder and discard it.</li>
	<li>For proper maintenance, replace the droplet reader oil and empty the waste receptacle as needed. Add 50 ml&#160;of 10% bleach to the waste bottle to prevent microbial growth.</li>
</ol>
2.4 DNA mutation profiling (as per the manufacturer&#8217;s recommended protocol)
Note: PCR-positive and PCR-negative droplets are counted to provide absolute quantification of target BRAF V600E DNA mutations in digital form, using data analysis software.
<ol>
	<li>Click Setup to enter information about the samples, assays, and experiments.</li>
	<li>In the Setup window, load a plate (filename.qlp), then click Analyze to open and analyze the data. The data analysis interface is separated into three windows: Results Table, Well Selector and Processed Data/Graphical Display.</li>
	<li>In the Processed Data window, concentration data for each target appear in the wells in the plate map and are tabulated in the Results Table. Click Concentration to visualize data in concentration plots.</li>
</ol>

<ol>
	<li>Vogelstein, B., 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=Genetic+alterations+during+colorectal-tumor+development.">Genetic alterations during colorectal-tumor development.</a> The New England journal of medicine. 319, 525-532 (1988).</li><li>Davies, H., 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=Mutations+of+the+BRAF+gene+in+human+cancer.">Mutations of the BRAF gene in human cancer.</a> Nature. 417, 949-954 (2002).</li><li>Solit, D. B., 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+mutation+predicts+sensitivity+to+MEK+inhibition.">BRAF mutation predicts sensitivity to MEK inhibition.</a> Nature. 439, 358-362 (2006).</li><li>Wong, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+anti-cancer+agents+targeting+the+Ras/Raf/+MEK/ERK+pathway.">Recent developments in anti-cancer agents targeting the Ras/Raf/ MEK/ERK pathway.</a> Recent patents on anti-cancer drug discovery. 4, 28-35 (2009).</li><li>Brose, M. 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. 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=High-Throughput+Droplet+Digital+PCR+System+for+Absolute+Quantitation+of+DNA+Copy+Number.">High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number.</a> Anal Chem. 83, 8604-8610 (1021).</li><li>Pinheiro, L. B., 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=Evaluation+of+a+Droplet+Digital+Polymerase+Chain+Reaction+Format+for+DNA+Copy+Number+Quantification.">Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification.</a> Anal Chem. 84, 1003-1011 (1021).</li><li>Jones, M., 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=Low+copy+target+detection+by+Droplet+Digital+PCR+through+application+of+a+novel+open+access+bioinformatic+pipeline,+'definetherain'.">Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, 'definetherain'.</a> Journal of virological methods. 202, 46-53 (2014).</li><li>Strain, M. 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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+detection+and+quantitation+of+DNA+copy+number+and+single+nucleotide+variants+with+single+color+droplet+digital+PCR.">High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR.</a> Anal Chem. 86, 2618-2624 (2014).</li><li>Bizouarn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+applications+using+digital+PCR.">Clinical applications using digital PCR.</a> Methods in molecular biology. 1160, 189-214 (2014).</li><li>McDermott, G. P., 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=Multiplexed+target+detection+using+DNA-binding+dye+chemistry+in+droplet+digital+PCR.">Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR.</a> Anal Chem. 85, 11619-11627 (2013).</li><li>Milbury, C. A., 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=Determining+lower+limits+of+detection+of+digital+PCR+assays+for+cancer-related+gene+mutations.">Determining lower limits of detection of digital PCR assays for cancer-related gene mutations.</a> Biomolecular detection and quantification. 1, 8-22 (2014).</li><li>Zhu, G., 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+Sensitive+Droplet+Digital+PCR+Method+for+Detection+of+EGFR+Activating+Mutations+in+Plasma+Cell-Free+DNA+from+Patients+with+Advanced+Non-Small+Cell+Lung+Cancer.">Highly Sensitive Droplet Digital PCR Method for Detection of EGFR Activating Mutations in Plasma Cell-Free DNA from Patients with Advanced Non-Small Cell Lung Cancer.</a> The Journal of molecular diagnostics: JMD. , (2015).</li><li>Fan, H., Gulley, M. 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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 ...

employing-digital-droplet-pcr-to-detect-braf-v600e-mutations-formalin

Research

JoVE Journal

Biology

13.2K vistas.  Seoul National University.  El objetivo de este video es demostrar cómo llevar a cabo la extracción de ADN automatizado de parafina embebido (FFPE) referencia las líneas celulares estándar fijado en formol y gotita digital de PCR (ddPCR) análisis para detectar mutaciones raras en un entorno clínico. La detección de mutaciones en muestras de FFPE demuestra la utilidad clínica de ddPCR en muestras FFPE. 

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

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.

We describe the use of quantitative immunoblotting to validate immunofluorescence histology coupled with image analysis as a means of quantifying a protein of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Our results demonstrate the utility of immunofluorescence histology for ascertaining the relative quantity of biomarker proteins in routine biopsy samples.

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An ...