We would like to thank L. Evans, B. Granger, D. Rissling, Z. Sim, A. Goodwin, K. Hayhurst and D. Kuch for assistance in the field. K. Galbreath kindly provided pika liver samples from the Bella Coola valley. Thanks to A. Gon&ccedil;alves da Silva and K. Larsen for interesting discussions that contributed to the design of this noninvasive genetic sampling method. This work was funded by the Natural Sciences and Engineering Research Council of Canada Discovery, and UBC Okanagan Individual Research grants to MAR. A Swiss National Science Foundation Doctoral Fellowship PBSKP3_128523 supported PH. This study was undertaken following the animal care protocol from the University of British Columbia (Certificate number: A07-0126).

1. Hair snare
Prior to setting up a snare, an ideal location has to be determined within the pika habitat or talus slope. This includes hay piles, which are vegetation caches that the animals collect in late summer as well as fresh scats found at latrine sites. Strips of packing tape (10-50cm in length) are rolled up to provide a 360&deg; sticky surface and are arranged in a web-like manner to envelope the entrance to the pika's hay pile (Fig. 1) or latrine site. Depending on the configuration of the rocks, a piece of fishing line may be used to support the structure of the hair snare, but this is often not necessary when entrances are quite small (&lt;30cm in diameter), a complete description of the use of fishing line can be found in Henry and Russello (2010)3. Hair snares are checked as often as possible, and hair samples deposited on the tape (Fig. 2) are then collected and labeled. Once transported back to the lab, hairs are removed from the sticky tape using sterile forceps and transferred into cryogenic tubes and stored at -20&deg;C until further manipulation. Hair samples clustered together along a hair snare are considered to belong to a single individual, while samples clustered independently are assumed to belong to different individuals. In the latter case, the hair is then placed in different tubes. These assumptions can later be tested by way of DNA fingerprints and calculations of probability of identity based on microsatellite genotypic data. 
2. DNA extraction
Since pika hair is very thin and contains a tiny root bulb, we have previously shown that a minimum of 25 hairs are required to obtain sufficient amounts of DNA for good quality downstream PCR amplification3. We used the DNA IQ system (Promega, Madison, WI, USA) and a slightly modified version of the manufacturer's instructions for DNA isolation from hair samples. Firstly, the hair sample is spun down in a micro-centrifuge in order to avoid loss of hair while opening the tube. Then the incubation solution is prepared by mixing 80 &mu;l of incubation solution with 10&mu;l of DTT (1M) and 10&mu;l of Proteinase K (18ng/ml) to result in a final concentration of 0.1 M DTT and 1.8ng/ml of Proteinase K. Incubation solution (100 &mu;l) is added to the sample and incubated at 56&deg;C for 1 hour. In the meantime, the lysis solution is prepared by adding 1 &mu;l of DTT (1M) for every 100 &mu;l of lysis buffer, resulting in a final DTT concentration of 0.01M. Prepared lysis solution (200 &mu;l) is then added to the sample, along with 7 &mu;l of DNA IQ resin, vortexed for 3 seconds at high speed and incubated at room temperature for 5 minutes. The sample is then vortexed for 2 seconds and inserted in the magnetic stand, where separation of magnetic beads from the solution occurs. The remaining solution is then aspirated and discarded, being careful not to disturb the pellet of magnetic resin. The sample is then treated with 100 &mu;l of the prepared lysis solution, vortexed and returned to the magnetic stand where separation occurs again. The lysis buffer is aspirated and discarded, and this step is repeated three times using a wash buffer (100 &mu;l). The sample is subsequently left to dry in the magnetic stand for 15 minutes. Once dry, 100 &mu;l of elution buffer is added to the sample and incubated at 65 &deg;C for 5 minutes. The sample is vortexed and inserted into the magnetic stand. The solution now containing the eluted DNA is then transferred to a 1.5 ml Eppendorf tube and stored at -20&deg;C until further manipulations. DNA from liver samples was also extracted using the DNA IQ system and used as a positive control for PCR amplifications.
3. PCR amplification
The DNA obtained from our noninvasive hair snare and liver samples were then used to amplify a suite of commonly used molecular markers (microsatellite, AFLP, mitochondrial cytochrome B and ZFX/ZFY sexing marker). PCRs were performed using a Veriti thermal cycler (Applied Biosystems, Foster City, CA, USA) in a 25 &mu;L volume containing: 10 - 20 ng DNA, 0.5 &mu;M of each primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 &mu;M dNTPs, 10 &mu; bovine serum albumin (BSA; New England Biolabs, Ipswich, MA, USA) and 0.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems). Cycling parameters for the microsatellite loci were optimised using a touchdown cycling program (10 min at 95&deg;C, 35 cycles at 95&deg;C for 30 s, 30 s annealing, and 45 s at 72&deg;C, followed by a final step at 72&deg;C for 10 min). The annealing temperature decreased by 1&deg;C per cycle from 60 to 55&deg;C until reaching the sixth cycle, at which point the 29 remaining cycles continued at 55&deg;C. Cycling parameters for the mitochondrial fragments were as described above but consisted of 35 cycles with an annealing temperature of 50&deg;C. Amplified Fragment Length Polymorphism was undertaken following the protocol for vertebrate genomes described by Bonin4. Lastly, molecular sexing was undertaken using the PCR-RFLP of ZFX/ZFY loci with HinfI restriction enzyme digestion5. The PCR products from the microsatellite, AFLPs and cytochrome B sequences were run onto a ABI 3130XL genetic analyser (Applied Biosystems), while digested ZFX/ZFX fragments were run alongside a 100 bp ladder (New England Biolabs) on a 3% agarose gel containing 2.5% SYBR Safe DNA gel stain (Invitrogen, Carlsbad, CA, USA) and visualised using a RED personal gel imaging system (Alpha Innotech, San Leandro, CA, USA). Microsatellite and AFLPs were visualized using Genemapper v3.7 (Applied Biosystems) and the mitochondrial DNA chromatogram was visualized using Sequencher v4.7 (Gene Code Corporation, Ann Harbour, MI, USA).
4. Representative results
The DNA extracted from hair samples obtained using our noninvasive snare were used to PCR amplify various type of molecular markers. As a basis for comparison, DNA extracted using liver samples was amplified alongside our hair samples. Nuclear microsatellite loci were successfully amplified for both hair and liver samples (Fig. 3). Although the intensity of the signal was greater when using the liver samples, this did not have an adverse effect on genotype scoring (Fig. 3). Similar results were achieved when using AFLPs (Fig. 4), mitochondrial DNA sequences (Fig. 5), and the ZFX/ZFY molecular sexing marker (Fig. 6).
<img src="/files/ftp_upload/2791/2791fig1.jpg" alt="Figure 1"/> 
Figure 1. An example of a noninvasive hair snare set up at a hay pile. Strips of rolled up clear packing tape are arranged in a web-like fashion to enclose the entrance to the hay pile. A piece of fishing line is used here to provide additional support for the hair snare.
<img src="/files/ftp_upload/2791/2791fig2.jpg" alt="Figure 2"/> Figure 2. A successful hair snare containing a large number of hair stuck to the packing tape.
<img src="/files/ftp_upload/2791/2791fig3.jpg" alt="Figure 3"/> 
Figure 3. A representative nuclear microsatellite chromatogram (Ocp6)8 as displayed in Genemapper v3.7 (Applied Biosystems). The top chromatogram was obtained by amplifying DNA from liver tissue, and is heterozygous (354/358) at this locus. The bottom chromatogram represents a different individual exhibiting a heterozygous genotype (358/362) based on DNA extracted from hair. While the signal of chromatogram from the hair sample has a lower intensity than the liver sample, genotype scoring is not adversely affected by this difference in signal.
<img src="/files/ftp_upload/2791/2791fig4.jpg" alt="Figure 4"/> 
Figure 4. A representative AFLP chromatogram (E31T32) as displayed in Genemapper v3.7 (Applied Biosystems). The top chromatogram was obtained by amplifying DNA from liver tissue, the bottom chromatogram represents the DNA extracted from hair. 
<img src="/files/ftp_upload/2791/2791fig5.jpg" alt="Figure 5"/> 
Figure 5. A representative mitochondrial DNA sequence chromatogram (cytochrome B) as displayed in Sequencher v4.7 (Gene Code Corporation). The top chromatogram was obtained by amplifying DNA from liver tissue, the bottom chromatogram represents the DNA extracted from hair.
<img src="/files/ftp_upload/2791/2791fig6.jpg" alt="Figure 6"/> 
Figure 6. A representative molecular sexing marker (ZFX/ZFY) amplified for both liver and hair samples. This approach relies on the observation that the Y chromosome possesses a HinfI restriction site in this region that the X chromosome lacks. Thus, females produce two co-migrating fragments of 432 bp while males produce fragments of 432 bp, 261 bp and 171 bp.

<ol>
	<li>Kendall, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demography+and+Genetic+Structure+of+a+Recovering+Grizzly+Bear+Population.">Demography and Genetic Structure of a Recovering Grizzly Bear Population.</a> J Wildlife Manage. 73, 3-3 (2009).</li><li>Rudnick, J. A., Katzner, T. E., Bragin, E. A., DeWoody, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-invasive+genetic+evaluation+of+population+size,+natal+philopatry,+and+roosting+behavior+of+non-breeding+eastern+imperial+eagles+(Aquila+heliaca)+in+central+Asia.">A non-invasive genetic evaluation of population size, natal philopatry, and roosting behavior of non-breeding eastern imperial eagles (Aquila heliaca) in central Asia.</a> Conserv. Genet. 9, 667-667 (2008).</li><li>Henry, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+population+structure+and+ex+situ+representation+of+the+endangered+Amur+tiger.">In situ population structure and ex situ representation of the endangered Amur tiger.</a> Mol Ecol. 18, 3173-3173 (2009).</li><li>Henry, P., Russello, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+high+quality+DNA+from+elusive+small+mammals+using+low-tech+hair+snares.">Obtaining high quality DNA from elusive small mammals using low-tech hair snares.</a> Eur J Wildlife Res. , (2010).</li><li>Bonin, A., Pompanon, F., Taberlet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+amplified+fragment+length+polymorphism+(AFLP)+markers+in+surveys+of+vertebrate+diversity.+Method+Enzymol+395.">Use of amplified fragment length polymorphism (AFLP) markers in surveys of vertebrate diversity. Method Enzymol 395.</a> , 145-145 (2005).</li><li>Fontanesi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexing+European+rabbits+(Oryctolagus+cuniculus),+European+brown+hares+(Lepus+europaeus)+and+mountain+hares+(Lepus+timidus)+with+ZFX+and+ZFY+loci.">Sexing European rabbits (Oryctolagus cuniculus), European brown hares (Lepus europaeus) and mountain hares (Lepus timidus) with ZFX and ZFY loci.</a> Mol Ecol Resour. 8, 1294-1294 (2008).</li><li>Piggott, M. P., Taylor, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+collection+of+animal+DNA+and+its+applications+in+conservation+management+and+understanding+the+population+biology+of+rare+and+cryptic+species.">Remote collection of animal DNA and its applications in conservation management and understanding the population biology of rare and cryptic species.</a> Wildl. Res. 30, 1-1 (2003).</li><li>Banks, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demographic+monitoring+of+an+entire+species+(the+northern+hairy-nosed+wombat,+Lasiorhinus+krefftii)+by+genetic+analysis+of+non-invasively+collected+material.">Demographic monitoring of an entire species (the northern hairy-nosed wombat, Lasiorhinus krefftii) by genetic analysis of non-invasively collected material.</a> Anim. Conserv. 6, 101-101 (2003).</li><li>Litvaitis, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+range-wide+survey+to+determine+the+current+distribution+of+New+England+cottontails.">A range-wide survey to determine the current distribution of New England cottontails.</a> Wildlife Soc B. 34, 1190-1190 (2006).</li><li>Peacock, M. M., Kirchoff, V. S., Merideth, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+nine+polymorphic+microsatellite+loci+in+the+North+American+pika,+Ochotona+princeps.">Identification and characterization of nine polymorphic microsatellite loci in the North American pika, Ochotona princeps.</a> Mol Ecol Notes. 2, 360-360 (2002).</li></ol>

No conflicts of interest declared.

Noninvasive genetic sampling has become an attractive alternative to traditional live trapping methods for several reasons. First, by unobtrusively collecting biological material (e.g., feces, hairs, feathers, saliva and mucus) from wild populations, researchers can study these species without disturbing, handling, or even observing them, thus reducing risks to both animals and researchers. Second, noninvasive genetic sampling enables biologists to study populations of elusive and rare species, a task that can prove difficult with more traditional live-trapping approaches 6. And third, NGS can potentially increase sample sizes by reducing disturbance to animals, sampling efforts, and costs, thus helping to minimise biases in estimates of population parameters 7. This latter point may prove crucial when dealing with threatened species, since biased estimates of population parameters may result in inappropriate management.
In the present video article we describe a simple, novel, inexpensive and noninvasive method to sample elusive small mammals, using the American Pika as an example. We demonstrate that DNA extracted from hair performs similarly to DNA extracted from liver samples with respect to microsatellite, AFLP, mitochondrial and sexing markers, making this noninvasive sampling method an attractive alternative to live trapping or lethal sampling. Overall, we anticipate that this method will be useful in the data collection stage of population genetic and behavioural studies of rare or elusive small mammal species, while minimizing impact on the organisms under study.

Hair snare:
<ul>
<li>Rolls of clear packing tape (3M, St. Paul, MN, USA)</li>
<li>Fishing line (10lb)</li>
<li>Cryogenic tube (2ml)</li>
<li>Sterile forceps</li>
<li>Liquid Nitrogen Dewar (optional)</li>
</ul>
DNA extraction:
<ul>
<li>DNA IQ System (Cat. # DC6701; Promega) with the tissue and hair extraction Kit (Cat. # DC6740; Promega).</li>
<li>MagneSphere Technology Magnetic Separation Stand (Cat. # Z5342; Promega)</li>
<li>Micro centrifuge</li>
<li>Hybridization oven or water bath</li>
<li>1.5 ml Eppendorf Tubes</li>
</ul>

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.

Watch this Scientific Journal Video about A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals at JoVE.com

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

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 ...

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20.6K ビュー.  University of British Columbia, Okanagan Campus. アメリカのナキウサギのために示されるように我々は、効率的にとらえどころのない小さな哺乳類から毛髪サンプルを収集するために非侵襲的サンプリングの手法を提示する。我々は、サンプリングされた髪の毛からDNAを抽出し、一般的に野生動物の生態と保全の研究で使用されている分子マーカーのいくつかのタイプを増幅することによってこの方法?...

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

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

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 ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...