Name: dna extraction from paraffin embedded material for genetic and epigenetic analyses
Uploaded: 2011-03-26T14:00:00
Duration: 13 min 32 s
Description: Watch this Scientific Journal Video about DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses at JoVE.com

We would like to thank members of the Lam lab for their evaluation and critiques of this video and article. This work was supported by funds from the Canadian Institutes for Health Research.

1. Procedural notes
<ul>
 <li>Xylene and phenol are toxic chemicals and should be handled in a fumehood. Refer to the material safety data sheet (MSDS) before use.</li>
 <li>Xylene dissolves certain plastics; polypropylene tubes should be used as they tolerate these compounds.</li>
 <li>Special tips may be purchased that contain an internal charcoal stopper. This prevents the xylene from dissolving any rubber components in the pipette. Alternatively, filter tips can be used.</li>
</ul>
2. Paraffin removal
<ol>
 <li>In a fumehood, add 800 &mu;l of xylene (VWR, CABDH6216-4) to the labeled tube containing your specimen and place on a rocker with gentle shaking for 5 to 15 minutes to dissolve the paraffin.</li>
 <li>Pellet the sample by spinning at max speed (14,000 rpm or 16 000 x g) in a microcentrifuge for 3 minutes. Carefully withdraw the xylene supernatant and dispose in a polypropylene tube for discard in the xylene waste. Be careful not to disturb the pellet.</li>
 <li>Repeat xylene wash steps 1 and 2until paraffin is fully dissolved. This usually requires two to three washes, depending on the size of the tissue sample. A fully dissolved sample will appear soft, and will sometimes lose its structural integrity. The integrity of the sample can be gently assessed with a pipette tip.</li>
</ol>
3. Ethanol rehydration
<ol>
 <li>Add 800 &mu;l of 100% ethanol (v/v) molecular biology grade ethanol, vortex, then spin for 3 minutes at 14,000 rpm in microcentrifuge. Remove the ethanol supernatant, being careful not to disturb the pellet.</li>
 <li>Add 800 &mu;l of 70% ethanol (100% (v/v) ethanol diluted in distilled water (dH2O)), vortex, and spin for 3 minutes at 14,000 rpm. Carefully remove the ethanol supernatant.</li>
 <li>Add 800 &mu;l of 50% ethanol (100% (v/v) ethanol diluted in distilled water (dH2O)), vortex, and spin for 5 minutes at 14,000 rpm. Carefully remove as much ethanol as possible by pipetting. Be extremely careful of disturbing the pellet at this point as fully rehydrated tissue will not pellet as well as dehydrated tissue.</li>
 <li>After removing the ethanol supernatant, air-dry the pellet for 5 minutes, being careful not to over dry.</li>
</ol>
4. Tissue digestion
<ol>
 <li>Add 200-500 &mu;l of Lysis Buffer (formula below). Re-suspend the pellet using a pipette tip or a vortex. Extra effort at this point to fully re-suspend the tissue will result in a more complete digestion of the sample and a better yield of DNA.</li>
 <li>Incubate samples at 56&deg;C in a water bath or heating block.</li>
 <li>For tissue cores, spike 20 &mu;l of proteinase K (20 mg/ml stock solution, Invitrogen, AM2548 ) morning and evening.</li>
 <li>Repeat above digestion steps for 2 to 5 days until the tissue is fully dissolved.</li>
</ol>
Lysis Buffer<ul>
<li> 10 mM Tris-HCl pH 8.0</li>
<li> 100 mM EDTA pH 8.0</li>
<li> 50 mM NaCl</li>
<li> 0.5% SDS</li>
<li> 200 &mu;g/ml proteinase K (add just before use)</li>
</ul>
5. DNA clean up
<ol>
 <li>Add an equal volume of buffer saturated phenol (Fisher, BP1750I-400) and mix by inversion. Spin for at least 5 minutes at 14,000 rpm in microcentrifuge.</li>
 <li>Transfer the aqueous layer to a new tube. Note the interphase: is there a lot of white material?</li>
 <li>Repeat steps 1 and 2 on the aqueous fraction until the interphase is clear (typically 3 or more times). Perform back extractions* when the interphase is fuzzy to increase final yield.</li>
 <li>Once the interphase is clear, add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) (Fisher, BP1752I-400) to the extracted aqueous fraction of your sample. Mix for 5 minutes and then spin for 5 minutes at 14,000 rpm. Thisreduces residual phenol and further sharpens the interphase, facilitating extraction of the aqueous layer</li>
 <li>Remove the aqueous layer to a new tube and treatwith RNase A at 100 &mu;g/ml for 1 hour at 37&deg;C.</li>
 <li>Repeat steps 1 to 4 to remove any remaining RNase A and collect the aqueous fraction. You should only need 1 or 2 buffer saturated phenol steps as there should be much less protein to remove than in the initial lysate.</li>
</ol>
*To perform back extractions add 50-100 &mu;l of dH20 to the sample tube containing the interphase and organic portion. Invert the tube to mix, and spin the sample at 14,000 rpm in a microcentrifuge for 5 minutes. Collect the aqueous phase and add it to the previously acquired aqueous extraction. Continue back extractions until the interphase is clear.
6. DNA precipitation
<ol>
 <li>Estimate the volume of your collected aqueous layer.</li>
 <li>Add 1/10 the volume of 3 M sodium acetate pH 5.2 and 1 volume of 100% isopropanol (v/v) molecular biology grade (or 2.5 volumes of 100% ethanol).</li>
 <li>Mix well and put on ice or in a -20&deg;C freezer for 30 minutes or overnight.</li>
 <li>Spin at maximum speed (14,000 rpm) at 4&deg;C in microcentrifuge for 10 minutes</li>
 <li>Discard the supernatant.</li>
 <li>Wash the pellet with 70% ice cold ethanol to remove unwanted salts.</li>
 <li>Re-suspend the pellet in the buffer of choice (usually dH2O).</li>
</ol>
7. DNA quantification
<ol>
 <li>Quantify the concentration of DNA. The A260:A280 ratio should be ~1.8. For small quantities of DNA, measurement using a NanoDrop spectrophotometer is preferred.</li>
 <li>Following quantification, gel electrophoresis should be performed to test the quality of your sample DNA and determine whether contaminating RNA has been completely degraded . </li>
 <li>High quality extracted DNA is now suitable for downstream applications, or can be stored at -20&deg;C for later use. If RNA is still present in your sample, repeat the DNA clean up and precipitation steps, and re quantify and qualify the quality of your samples.</li>
</ol>

<ol>
	<li>Santos, M. C., Saito, C. P., Line, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+genomic+DNA+from+paraffin-embedded+tissue+sections+of+human+fetuses+fixed+and+stored+in+formalin+for+long+periods.">Extraction of genomic DNA from paraffin-embedded tissue sections of human fetuses fixed and stored in formalin for long periods.</a> Pathol Res Pract. 204, 633-636 (2008).</li><li>Hilz, H., Wiegers, U., Adamietz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+proteinase+K+action+by+denaturing+agents:+application+to+the+isolation+of+nucleic+acids+and+the+degradation+of+'masked'+proteins.">Stimulation of proteinase K action by denaturing agents: application to the isolation of nucleic acids and the degradation of 'masked' proteins.</a> Eur J Biochem. 56, 103-108 (1975).</li><li>Sambrook, J. o. s. e. p. h., R, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , (2001).</li><li>Kennett, J. Y., Watson, S. K., Saprunoff, H., Heryet, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+demonstration+of+whole+genome+array+comparative+genomic+hybridization.">Technical demonstration of whole genome array comparative genomic hybridization.</a> J Vis Exp. , (2008).</li><li>Thu, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylated+DNA+immunoprecipitation.">Methylated DNA immunoprecipitation.</a> J Vis Exp. , (2009).</li><li>Tang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+extraction+from+formalin-fixed,+paraffin-embedded+tissue.">DNA extraction from formalin-fixed, paraffin-embedded tissue.</a> Cold Spring Harb Protoc. , (2009).</li><li>Hongxin Fan, M. L. G., G, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Extraction+from+Paraffin-Embedded+Tissues.">DNA Extraction from Paraffin-Embedded Tissues.</a> Molecular Pathology Protocols. 49, 1-4 (2001).</li></ol>

No conflicts of interest declared.

Biopsied or surgically excised tissues for histopathologic analysis and diagnosis are often formalin fixed and paraffin embedded (FFPE) for long term storage. With the growing interest in understanding the genetic basis of disease, the ability to extract DNA from these samples represents an invaluable source of diagnostic material that can be used for genomic analysis and translational studies. Historically FFPE samples were not considered a viable source for molecular analysis as nucleic acids may be heavily modified by protein-nucleic acid and protein-protein cross linking6. However, the discovery that protease digestion releases fragmented nucleic acids which are suitable for downstream analyses including PCR, array CGH, sequencing and methylation profiling, enables the use of these valuable specimens for genetic analysis 6.
DNA extraction from paraffin-embedded tissues is a robust procedure that relies on differential solubility to purify DNA. Extracted DNA quality and quantity and the success of subsequent DNA amplification is dependent on a number of parameters before, during and after extraction. These include, but are not limited to: type and amount of tissue, the type of fixative used for tissue preservation, the duration of fixation, age of the paraffin block and storage conditions, as well as the length of the desired DNA segment to be amplified 1,7. Removal of paraffin from the tissue is the most critical step for successful extraction as undissolved paraffin leads to poor sample quality and inhibition of PCR amplification.

<table border="1">

	<tbody>
 <tr>	
 	<th>Name</th>
 <th>Company</th>
 <th>Catalogue Number</th>
 <th>Comments</th>
 </tr>
 <tr>	
 	<td>Xylene</td>
 <td>VWR</td>
 <td>CABDH6216- 4</td>
 <td>-</td>
 </tr>
		<tr>	
 	<td>1.7 ml SafeSeal Microcentrifuge Tubes</td>
 <td>Sorenson BioScience</td>
 <td>11510</td>
 <td>-</td>
 </tr>
 <tr>	
 	<td>Spectrafuge 16M Microcentrifuge</td>
 <td>Labnet</td>
 <td>C0160-B</td>
 <td>-</td>
 </tr>
		<tr>	
 	<td>Lysis Buffer</td>
 <td>-</td>
 <td>-</td>
 <td>10 mM Tris-HCl pH8, 
100 mM EDTA pH 8, 
50 mM NaCl, 
0.5% SDS, 
200 &mu;g/ml Proteinase K
</td>
 </tr>
		<tr>	
 	<td>Proteinase K Stock Solution</td>
 <td>Invitrogen</td>
 <td>AM2548</td>
 <td>20 mg/ml</td>
 </tr>
 <tr>	
 	<td>Proteinase K Stock Solution</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
		<tr>	
 	<td>Buffer Saturated Phenol pH 6.6/7.9</td>
 <td>Fisher</td>
 <td>BP1750I-400</td>
 <td>-</td>
 </tr>
 <tr>	
 	<td>Phenol: chloroform: Isoamylalcohol (25:24:1, pH 6.7/8.0)</td>
 <td>Fisher</td>
 <td>BP1752I-400</td>
 <td>-</td>
 </tr>
		<tr>	
 	<td>RNase A</td>
 <td>Roche</td>
 <td>10109169001</td>
 <td>100mg</td>
 </tr>

		<tr>	
 	<td>3M sodium Acetate pH 5.2</td>
 <td>-</td>
 <td>-</td>
 <td>40.81g NaOAc in 80ml 
Adjust to pH 5.2 with glacial acetic acid 
Add dH2O to 100ml 
Autoclave
		</td>
 </tr>
		<tr>	
 	<td>ND 3300 Spectrophotometer</td>
 <td>NanoDrop</td>
 <td>-</td>
 <td>-</td>
 </tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses at JoVE.com

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

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 ...

dna-extraction-from-paraffin-embedded-material-for-genetic-epigenetic

Research

JoVE Journal

Biology

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

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 ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

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 ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

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 ...

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

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 ...