Name: extraction and analysis of cortisol from human and monkey hair
Uploaded: 2014-01-24T08:45:00
Description: Watch this Scientific Journal Video about Extraction and Analysis of Cortisol from Human and Monkey Hair at JoVE.com

We thank Kymberlee O&#39;Brien, Celia Moore, and Edward Tronick (Department of Psychology, University of Massachusetts,&#160;Boston) for providing the human hair samples analyzed in this study, and Stephen Suomi and Amanda Dettmer (Laboratory of Comparative Ethology, NICHD) for providing the rhesus monkey hair samples. Initial development and continued use of this method has been supported by NIH RR11122 to M.A.N.

1. Sample Collection and Storage
<ol>
	<li>Human hair
	<ol>
		<li>Secure the entire length of the hair to be sampled (up to a pencil-width in diameter) with a rubber band or clip. Cut hair as close to the scalp as possible (taking care not to nick the skin) with a clean scissors. Note: The standard sampling area is the posterior vertex of the skull.</li>
		<li>Some investigators have reported a decline in human hair CORT levels&#160; with distance from the scalp6, which may be due to washout from repeated ex...

<ol>
	<li>Selye, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+the+general+adaptation+syndrome.">Stress and the general adaptation syndrome.</a> Br. Med. J. 1 (4667), 1383-1392 (1950).</li><li>Meyer, J. S., Novak, 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=Minireview:+Hair+cortisol:+A+novel+biomarker+of+hypothalamic-+pituitary-adrenocortical+activity.">Minireview: Hair cortisol: A novel biomarker of hypothalamic- pituitary-adrenocortical activity.</a> Endocrinology. 153, 4120-4127 (2012).</li><li>Tiefenbacher, S., Novak, M. A., Lutz, C. K., Meyer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+and+neurochemistry+of+self-injurious+behavior:+A+nonhuman+primate+model.">The physiology and neurochemistry of self-injurious behavior: A nonhuman primate model.</a> Front. Biosci. 10, 1-11 (2005).</li><li>Davenport, M. D., Tiefenbacher, S., Lutz, C. K., Novak, M. A., Meyer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+endogenous+cortisol+concentrations+in+the+hair+of+rhesus+macaques.">Analysis of endogenous cortisol concentrations in the hair of rhesus macaques.</a> Gen. Comp. Endocrinol. 147, 255-261 (2006).</li><li>Davenport, M. D., Lutz, C. K., Tiefenbacher, S., Novak, M. A., Meyer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rhesus+monkey+model+of+self+injury:+Effects+of+relocation+stress+on+behavior+and+neuroendocrine+function.">A rhesus monkey model of self injury: Effects of relocation stress on behavior and neuroendocrine function.</a> Biol. Psychiatry. 63, 990-996 (2008).</li><li>Kirschbaum, C., Tietze, A., Skoluda, N., Dettenborn, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+as+a+retrospective+calendar+of+cortisol+production+-+Increased+cortisol+incorporation+into+hair+in+the+third+trimester+of+pregnancy.">Hair as a retrospective calendar of cortisol production - Increased cortisol incorporation into hair in the third trimester of pregnancy.</a> Psychoneuroendocrinology. 34, 32-37 (2009).</li><li>Hamel, A. F., Meyer, J. S., Henchey, E., Dettmer, A. M., Suomi, S. J., Novak, 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=Effects+of+shampoo+and+water+washing+on+hair+cortisol+concentrations.">Effects of shampoo and water washing on hair cortisol concentrations.</a> Clin. Chim. Acta. 412, 382-385 (2011).</li><li>Manenschijn, L., Koper, J. W., Lamberts, S. W., van Rossum, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+method+to+measure+long+term+cortisol+levels.">Evaluation of a method to measure long term cortisol levels.</a> Steroids. 76, 1032-1036 (2011).</li><li>Thomson, S., Koren, G., Fraser, L. A., Rieder, M., Friedman, T. C., Van Uum, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+analysis+provides+a+historical+record+of+cortisol+levels+in+Cushing&#8217;s+syndrome.">Hair analysis provides a historical record of cortisol levels in Cushing&#8217;s syndrome.</a> Exp. Clin. Endocrinol. Diabetes. 118, 133-138 (2010).</li><li>LeBeau, M. A., Montgomery, M. A., Brewer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+variations+in+growth+rate+and+sample+collection+on+interpreting+results+of+segmental+analyses+of+hair.">The role of variations in growth rate and sample collection on interpreting results of segmental analyses of hair.</a> Forensic Sci. Int. 210, 110-116 (2011).</li><li>Dolnick, E. H., Montagna, W., Dobson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+of+hair+growth+in+Macaca+mulatta.">Variability of hair growth in Macaca mulatta.</a> Adv. Biol. Skin. IX, 121-128 (1969).</li><li>Sauv&#233;, B., Koren, G., Walsh, G., Uum Tokmakejian, S. V. a. n., H, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cortisol+in+human+hair+as+a+biomarker+of+systemic+exposure.">Measurement of cortisol in human hair as a biomarker of systemic exposure.</a> Clin. Invest. Med. 30, 183-191 (2007).</li><li>Gow, R., Thomson, S., Rieder, M., Van Uum, S., Koren, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assessment+of+cortisol+analysis+in+hair+and+its+clinical+applications.">An assessment of cortisol analysis in hair and its clinical applications.</a> Forensic Sci. Int. 196, 32-37 (2010).</li><li>Bechsh&#248;ft, T. &. #. 2. 1. 6. ;., Sonne, C., Dietz, R., Born, E. W., Novak, M. A., Henchey, E., Meyer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortisol+levels+in+hair+of+East+Greenland+polar+bears.">Cortisol levels in hair of East Greenland polar bears.</a> Sci. Total Environ. 409, 831-834 (2011).</li><li>Cooper, G. A. A., Kronstrand, R., Kintz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Society+of+Hair+Testing+guidelines+for+drug+testing+in+hair.">Society of Hair Testing guidelines for drug testing in hair.</a> Forensic Sci. Int. 218, 20-28 (2012).</li><li>Ito, N., Ito, T., Kromminga, A., Bettermann, A., Takigawa, M., Kees, F., Straub, R. H., Paus, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hair+follicles+display+a+functional+equivalent+of+the+hypothalamic-pituitary-adrenal+(HPA)+axis+and+synthesize+cortisol.">Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal (HPA) axis and synthesize cortisol.</a> FASEB J. 19, 1332-1334 (2005).</li><li>Russell, E., Koren, G., Rieder, M., Van Uum, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cortisol+as+a+biological+marker+of+chronic+stress:+Current+status,+future+directions+and+unanswered+questions.">Hair cortisol as a biological marker of chronic stress: Current status, future directions and unanswered questions.</a> Psychoneuroendocrinology. 37, 589-601 (2012).</li><li>Stalder, T., Kirschbaum, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cortisol+in+hair+-+State+of+the+art+and+future+directions.">Analysis of cortisol in hair - State of the art and future directions.</a> Brain Behav. Immun. 26, 1019-1029 (2012).</li><li>Staufenbiel, S. M., Penninx, B. W., Spijker, A. T., Elzinga, B. M., van Rossum, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cortisol,+stress+exposure,+and+mental+health+in+humans:+A+systematic+review.">Hair cortisol, stress exposure, and mental health in humans: A systematic review.</a> Psychoneuroendocrinology. 38, 1220-1235 (2013).</li></ol>

The hair CORT procedure described above is simple to perform, is relatively inexpensive, makes use of readily available chemicals, reagents, and supplies, and requires equipment that, with one exception, is likely to be present in a typical analytical laboratory. The exception is a grinding apparatus such as a ball mill or mini-beadbeater. We note that some research groups mince hair samples into small fragments roughly 1&#160;mm in length12, but based on our observations we recommend performing grinding inste...

Measurement of CORT in plasma, saliva, or occasionally in urine or feces has been used as an index of physiological stress since Selye&#39;s discovery of the role of the HPA axis in stress1. Although numerous papers have been published relating HPA activity to acutely stressful situations, the field has been hampered by the lack of a simple and reliable index of chronic physiological stress. This problem arises because plasma and saliva both yield &#34;point&#34; estimates of HPA activity that are subject to circadian variation and can be confounded by environmental disturbances. Urinary and fecal samples yield measurements of CORT and/or metabolite excretion that span a number of hours up to a full day in some cases. Collection of multiple samples using any of these matrices may provide a rough composite index of CORT levels over time; however, none of these approaches provides a truly long-term index of HPA activity and the responsiveness of this system to chronic stressors.
Measuring CORT in hair has begun to fill this important need in the stress literature. Initial studies by several laboratories demonstrated the presence of CORT in human hair but did not investigate whether hair CORT levels changed as a function of stress2. As our laboratory has been interested for many years in the regulation of the rhesus monkey HPA axis by various social and behavioral factors3, we set out to establish and validate methods for extraction and analysis of rhesus monkey hair4. Based on the premise that blood-borne CORT is slowly and continuously incorporated into growing hair, the purpose of this new method was to use levels of hair-derived CORT as an integrated index of HPA activity over periods of weeks to months.
Several methodological challenges were encountered in developing the present protocol. First, previous studies had shown that small amounts of circulating CORT are excreted in sweat and sebum and therefore could coat the outside of the hair shaft2. In order to eliminate this potential confound, we developed a mild wash procedure that appears to remove external CORT while having a minimal effect on CORT present within the growing hair shaft. Thus, monkey hair subjected to this procedure (i.e.&#160;two 3-min washes with isopropanol) lost approximately 7-8% of the total hair CORT content, and a third wash removed less than 1% more steroid from the sample4. There appears to be more external CORT in human hair, since the same procedure removed an average of 27% total CORT content from the samples (K. Rosenberg and J. Meyer, unpublished). Like monkey hair, however, an additional wash contained much less CORT (about 7%) than the first two washes. Therefore, results from both monkey and human hair support the contention that most (if not all) external CORT can be removed while maintaining a major fraction of CORT within the internal hair matrix. Second, our pilot studies also showed that grinding the hair prior to extraction significantly increased CORT recovery from the sample, presumably by breaking open the complex proteinaceous matrix of the hair shaft as well as increasing the surface area available for solvent penetration. Two different grinding methods were developed, each with advantages and disadvantages. Method 1, which uses a ball mill, has the advantage of producing the finest powder. However, a ball mill is a relatively expensive equipment item and, if used with standard grinding jars and balls, it is capable of grinding only two samples at a time. Small samples are also difficult to process using a ball mill with standard grinding jars. Method 2, which uses a beadbeater, is less effective in its grinding ability. As a result, average CORT recovery is approximately 10% lower using this method compared to the ball mill (unpublished data). On the other hand, a beadbeater is considerably less expensive than a ball mill, 16-24 samples can be ground at once depending on the model, and the method is well suited for small samples. Because of the above mentioned differential recovery, it is advisable to use the same grinding method for all samples within a particular study.
Once the hair samples have been processed, they are extracted with methanol and CORT in the extracts is analyzed by means of a sensitive and specific commercial EIA kit originally designed to measure salivary CORT. The extraction and assay procedures were validated in part by demonstrating that serial dilutions of extracts from monkey hair samples yielded EIA readings that closely paralleled the readings obtained from authentic CORT standards. We then showed that hair CORT (in addition to plasma and salivary CORT) was sensitive to the major life stressor of an administratively mandated relocation of the monkeys to new housing quarters4,5. The present paper provides a detailed account of the methods used routinely in our laboratory to process human and monkey hair samples and to extract and analyze CORT from such samples.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">HPLC-grade isopropanol</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:119px;">A451</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">HPLC-grade methanol</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:119px;">A452</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Salivary cortisol assay kits</td>
			<td style="width:116px;">Salimetrics</td>
			<td style="width:119px;">1-3002</td>
			<td>See manufacturer&#39;s kit insert for information on assay sensitivity and specificity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-ml polypropylene screw-cap centrifuge tubes</td>
			<td style="width:116px;">Max Scientific</td>
			<td>10-9151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">1.5-ml Safe-Lock microcentrifuge tubes</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:119px;">05-402-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">2.0-ml Safe-Lock microcentrifuge tubes</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:119px;">05-402-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.0-ml XXTuff reinforced microvials</td>
			<td style="width:116px;">BioSpec</td>
			<td>330TX</td>
			<td>Use with mini-beadbeater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.2-mm chrome-steel beads</td>
			<td style="width:116px;">BioSpec</td>
			<td>11079132c</td>
			<td>Use with mini-beadbeater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">10-ml stainless steel grinding jars</td>
			<td style="width:116px;">Retsch</td>
			<td style="width:119px;">02.462.0061</td>
			<td>Use with mixer mill</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-mm stainless steel grinding balls</td>
			<td style="width:116px;">Retsch</td>
			<td style="width:119px;">05.368.0037</td>
			<td>Use with mixer mill</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Savant activated carbon cartridge</td>
			<td style="width:116px;">Fisher</td>
			<td>DTK120R</td>
			<td>Use with Savant chemical trap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;"></td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;"></td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Name of Equipment</td>
			<td style="width:116px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Rotator for 15-ml centrifuge tubes</td>
			<td style="width:116px;">Fisher</td>
			<td>S02135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Rotator for microcentrifuge tubes</td>
			<td style="width:116px;">Fisher</td>
			<td>NC9854190&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benchtop centrifuge for microcentrifuge tubes</td>
			<td style="width:116px;">Fisher</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">MM 200 mixer mill</td>
			<td style="width:116px;">Retsch</td>
			<td style="width:119px;">20.746.0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-Beadbeater 16&#160;</td>
			<td style="width:116px;">BioSpec</td>
			<td>607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Savant DNA Speedvac</td>
			<td style="width:116px;">Fisher</td>
			<td>DNA120-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Savant refrigerated vapor trap</td>
			<td style="width:116px;">Fisher</td>
			<td>RVT400-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Savant chemical trap</td>
			<td style="width:116px;">Fisher</td>
			<td>SCT120</td>
			<td>Alternative to refrigerated vapor trap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate reader</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate washer</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate mixer</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Multichannel pipetter</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Analytical balance</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction and analysis of cortisol from human and monkey hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Figure 1&#160;shows the printout from a representative set of human hair samples (adult male and female human subjects) processed using method 2 grinding and extraction. Computer software was used to generate the data output and to fit a 4-parameter sigmoidal curve to the CORT standards (Figure 2). The between-well CVs from this plate ranged from 0.01-5.73% with an average intra-assay CV of 1.34%. The inter-assay CV determined using the QC values from nine recent human hair assays was 4....

Watch this Scientific Journal Video about Extraction and Analysis of Cortisol from Human and Monkey Hair at JoVE.com

Extraction and Analysis of Cortisol from Human and Monkey Hair

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

extraction-and-analysis-of-cortisol-from-human-and-monkey-hair

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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 ...

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 ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

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 ...

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 ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on host and/or pathogen chromatin. Chromatin preparation from tissues creates additional challenges that need to be overcome to obtain reproducible and reliable protocols comparable to those used for cell culture. Tissue disruption and fixation are critical steps to achieve efficient shearing of chromatin. Coexistence of different cell types and clusters may also require different shearing times to reach optimal fragment size and hinders shearing reproducibility. The purpose of this method is to achieve reliable and reproducible host chromatin preparations from frozen tissue (liver) suitable for both ChIP-qPCR and next generation sequencing (NGS) applications. We observed that the combination of liquid nitrogen tissue pulverization followed by homogenization leads to increased reproducibility compared to homogenization only, since it provides a suspension consisting mostly of dissociated single cells that can be efficiently sheared. Moreover, the fixation step should be performed under mild rotation to provide homogeneous crosslinking. The fixed material is then suitable for buffer-based nuclei isolation, to reduce contamination of cytoplasmic protein and pathogen DNAs and RNAs (when applicable), avoiding time-consuming centrifugation gradients. Subsequent sonication will complete nuclear lysis and shear the chromatin, producing a specific size range according to the chosen shearing conditions. The size range should fall between 100 and 300 nt for NGS applications, while it could be higher (300-700 nt) for ChIP-qPCR analysis. Such protocol adaptations can greatly improve chromatin analyses from frozen tissue specimens.

This protocol focuses on chromatin preparation from snap frozen tissues and it is suitable for Crosslinking Chromatin Immunoprecipitation (X-ChIP) followed by either quantitative PCR analysis (X-ChIP-qPCR) or next generation sequencing approaches (X-ChIP-seq).

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...