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><tbody><tr><td>Xylene</td><td>VWR international</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 International</td><td>C0160-B</td><td>-</td></tr><tr><td>Lysis Buffer</td><td></td><td></td><td>10 mM Tris-HCl pH8,&lt;br/&gt;100 mM EDTA pH 8,&lt;br/&gt;50 mM NaCl,&lt;br/&gt;0.5% SDS,&lt;br/&gt;200 &amp;#x03BC;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></td><td></td><td></td></tr><tr><td>Buffer Saturated Phenol pH 6.6/7.9</td><td>Fisher Scientific</td><td>BP1750I-400</td><td>-</td></tr><tr><td>Phenol: chloroform: Isoamylalcohol (25:24:1, pH 6.7/8.0)</td><td>Fisher Scientific</td><td>BP1752I-400</td><td>-</td></tr><tr><td>RNase A</td><td>Roche Group</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&lt;br/&gt;Adjust to pH 5.2 with glacial acetic acid&lt;br/&gt;Add dH&lt;sub&gt;2&lt;/sub&gt;O to 100ml&lt;br/&gt;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

56.3K Views.  BC Cancer Research Centre. Deze video toont het protocol voor DNA-extractie van formaline gefixeerde in paraffine ingebedde materiaal. Dit is een multi-dagen procedure waarbij weefsel secties worden ontdaan met xyleen, gerehydrateerd met ethanol en behandeld met proteinase K te zuiveren en DNA te isoleren voor latere gen-specifieke of genome-wide analyse.

Video: DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Neuroscience

Optimized High Quality DNA Extraction from Formalin-Fixed Paraffin-Embedded Human Atherosclerotic Lesions

Formalin-fixed paraffin-embedded (FFPE) tissues represent a valuable source for molecular analyses and clinical genomic studies. These tissues are often poor in cells or difficult to process. Therefore, nucleic acids need to be carefully isolated. In recent years, various methods for DNA isolation have been established for tissues from many diseases, mostly cancer. Unfortunately, genomic DNA extracted from FFPE tissues is highly degraded due to the cross-linking between nucleic acid strands and proteins, as well as random breakings in sequence. Therefore, DNA quality from these samples is markedly reduced, making it a challenge for further molecular downstream analyses. Other problems with difficult tissues are, for example, the lack of cells in calcified human atherosclerotic lesions and fatty tissue, small skin biopsies, and consequently low availability of the desired nucleic acids as it is also the case in old or fixed tissues.
In our laboratories, we have established a method for DNA extraction from formalin-fixed atherosclerotic lesions, using a semi-automated isolation system. We compared this method to other commercially available extraction protocols and focused on further downstream analyses. Purity and concentration of the DNA were measured by spectrometry and fluorometry. The degree of fragmentation and overall quality were assessed.
The highest DNA quantity and quality was obtained with the modified blood DNA protocol for the automated extraction system, instead of the commercial FFPE protocol. With this step-by-step protocol, DNA yields from FFPE samples were in average four times higher and fewer specimens failed the extraction process, which is critical when dealing with small-vessel biopsies. Amplicon sizes from 200&#8211;800 bp could be detected by PCR. This study shows that although DNA obtained from our FFPE tissue is highly fragmented, it can still be used for successful amplification and sequencing of shorter products. In conclusion, in our hands, the automated technology appears to be the best system for DNA extraction, especially for small FFPE tissue specimen.

Here, we present a protocol for semi-automated DNA extraction from formalin-fixed paraffin-embedded lesions of human carotid arteries. The tissue lysis is performed without toxic xylene, which is followed by an automated DNA extraction protocol, including a second lysis step, binding of DNA to paramagnetic particles for cellulose based binding, washing steps, and DNA elution.

Formalin-fixed paraffin-embedded (FFPE) tissues represent a valuable source for molecular analyses and clinical genomic studies. These tissues are often ...

Genetics

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

Behavior

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Immunology and Infection

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

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 ...

Enhancing Tumor Content through Tumor Macrodissection

The presence of contaminating non-tumor tissues in formalin-fixed paraffin-embedded (FFPE) tissues can greatly undermine genomic studies. Herein we describe macrodissection, a method designed to augment the percentage tumor content of a tissue specimen by removing and eliminating unwanted tissue prior to performing downstream nucleic acid extractions. FFPE tissue blocks were sectioned to produce 4-5 &#181;m slide-mounted tissue sections. A representative section was submitted for hematoxylin and eosin (H&#38;E) staining and subsequently reviewed by a board-certified pathologist. During the review, the pathologist identified and marked the regions of tumor tissue in the H&#38;E. Once complete, the demarked H&#38;E was used to guide resection of the serial unstained sections from the same tissue block. To demonstrate the effects of macrodissection, RNA extracted from matched macrodissected and non-dissected Diffuse Large B-Cell Lymphomas (DLBCL) were run on a digital gene expression assay capable of determining DLBCL subtype and BCL2 translocation status. The results showed that macrodissection changed the subtype or BCL2 translocation status calls in 60% of the samples examined. In conclusion, macrodissection is a simple and effective method for performing tumor enrichment prior to nucleic acid extractions, the product of which can then be confidently used in downstream genomic studies.

This protocol presents a method to increase the percent tumor content of formalin-fixed paraffin-embedded tissue samples.

The presence of contaminating non-tumor tissues in formalin-fixed paraffin-embedded (FFPE) tissues can greatly undermine genomic studies. Herein we ...

Deparaffinization and Rehydration of Tissue Sections: A Two-step Procedure to Remove Paraffin Wax from FFPE Tissue Slides Followed by Tissue Rehydration

Source: Sugimoto, K. et al. <a href="https://www.jove.com/video/61355" target="_blank">Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the processing of FFPE or formalin-fixed paraffin-embedded sections obtained from gastrointestinal tumor tissue. The deparaffinization and rehydration steps allow the processing of the tissue specimen for subsequent analytical applications.

Source: Sugimoto, K. et al. Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer. J. Vis. Exp. (2020)
In this video, we demonstrate the ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

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