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

우리는이 연구에서 분석 사람의 모발 샘플을 제공 Kymberlee 오브라이언, 셀리아 무어, 에드워드 Tronick 씨 (심리학과, 매사 추세 츠, 보스턴 대학교) 감사, 스티븐 개가 아만다 D​​ettmer (비교 행동학, NICHD의 실험)에 대한 붉은 털 원숭이의 머리 샘플을 제공한다. 초기 개발 및이 방법을 계속 사용 MAN에 NIH RR11122에 의해 지원 된

1. 시료 채취 및 보관 <ol><li> 사람의 모발 <ol><li> 고무 밴드 나 클립 (직경 연필 폭까지) 샘플링 할 수있는 머리의 전체 길이를 고정합니다. 최대한 두피에 가깝게 잘라 머리 깨끗한 가위 (닉은 피부를 돌보는). 주 : 표준 샘플링 에리어가 두개골의 후방 정점이다. </li><li> 일부 연구자들은 비록 볼 Manenschijn 등. 8 (물과 샴푸 7 반복 노출에서 유실로 인해 수 있습니다 두피 6?...

<ol>
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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>

전술 한 헤어 CORT 절차 수행하도록 심플 상대적으로 저렴하고, 쉽게 이용할 화학 시약 및 소모품의 사용을하게하고, 한 가지 예외 전형적인 분석 실험실에 존재할 가능성이 있다고, 장비를 필요로한다. 예외는 볼밀 미니 beadbeater 같은 연마 장치이다. 우리는 몇 가지 연구 그룹은 작은 조각 길이 12 대략 1mm에 머리 샘플을 말하다 있습니다,하지만 우리는 연삭 수행하는 대신 가능하면 닦지 ?...

혈장, 타액, 또는 때때로 소변이나 대변의 CORT 측정 스트레스 1 HPA 축의 역할 Selye 디스커버리시기 생리적 스트레스의 지표로서 사용되었다. 수많은 논문이 급성 스트레스 상황에 HPA 활동과 관련된 발행되었지만, 필드는 만성 생리적 스트레스 간단하고 신뢰성 지수의 부족에 의해 방해되었다. 이 문제가 발생주기 차이가있을 수있는 환경 교란에 의해 혼동 될 수 HPA 활동의 플라즈마 침 수율 모두 &quot;포인트&quot;예상하기 때문이다. 오줌과 배설물 샘플 어떤 경우에는 하루 종일까지 시간의 숫자에 걸쳐 CORT 및 / 또는 대사 산물의 배설의 측정 값을 얻을 수 있습니다. 그러나, 이들 접근법 중 어느 것도 HPA 활성의 진정한 장기 인덱스 및이 싸이의 응답 성을 제공하지 않는다; 이러한 행렬들의 임의 시간 경과 CORT 레벨의 거친 지수를 제공 할 수있다하여 여러 샘플의 컬렉션만성 스트레스에 줄기. 머리에 CORT를 측정하는 것은 스트레스 문학이 중요한 필요를 채우기 위해 시작했다. 여러 실험실에 의한 초기 연구는 인간의 머리에 CORT의 존재를 증명하지만, 머리 CORT 수준이 스트레스 2의 함수로 변경 여부를 조사하지 않았다. 우리 연구실은 다양한 사회 및 행동 요인 (3)에 의해 붉은 털 원숭이 HPA 축 조절에 몇 년 동안 관심이있다, 우리는 붉은 털 원숭이 머리 4의 추출 및 분석을위한 방법을 설정하고 확인하기 위해 밖으로 설정합니다. 혈액 유래 CORT 천천히 연속적으로 성장하는 모발에 혼입되는 것을 전제로,이 새로운 방법의 목적은 개월 주내 걸쳐서 HPA 활성의 통합 지표로서 헤어 유래 CORT 수준을 사용하는 것이었다. 몇 가지 방법 론적 문제는 본 프로토콜 개발에 발견되었습니다. 첫째, 이전의 연구는 적은 양의 O를 표시했다F 땀과 피지에 배설 CORT을 순환하기 때문에 코트 머리 축 (2)의 외부를 할 수 있습니다. 이러한 잠재적 혼동을 제거하기 위해, 우리는 성장 모간 내에 존재 CORT에 최소한의 효과를 가지면서 외부 CORT 제거 나타나는 온화한 세척 과정을 개발 하였다. 따라서,이 절차 (이소프로판올, 즉 두 개의 3 분 세척) 실시 원숭이 머리 전체 머리 CORT 내용의 약 ~ 8 %를 손실 및 제 3 세척 샘플 4에서 1 % 미만의 더 많은 스테로이드를 제거했습니다. 같은 절차 (게시되지 않은 K. 로젠버그와 J. 마이어) 샘플에서 27 % 총 CORT 콘텐츠의 평균을 제거하기 때문에, 사람의 모발에 더 많은 외부 CORT이있을 나타납니다. 원숭이 머리를 좋아하지만, 추가 세척은 처음 두 세척보다 훨씬 적은 CORT (약 7 %)가 포함되어 있습니다. 따라서, 원숭이 및 사람의 모발 양의 결과가 주요 골절을 유지하면서, 대부분 (전부는 아니더라도) 외부 CORT가 제거 될 수 있다는 논쟁을 지원내부 헤어 매트릭스 내에서 CORT의 기. 둘째, 우리의 파일럿 연구는 또한 보여 주었다 아마도 모간의 복합 단백질 성 매트릭스를 오픈 침입뿐만 아니라 용매의 침투 가능한 표면 면적을 증가시킴으로써, 샘플에서 유의하게 증가 COR​​T 회수를 추출하여 모발 전에 연삭. 두 개의 서로 다른 연삭 방법은 장점과 단점을 각각 개발되었다. 볼 밀을 사용하여 방법 1, 각종 분말을 제조하는 장점을 갖는다. 그러나, 볼 밀은 비교적 고가의 장비 아이템이며 표준 연삭 항아리와 볼을 사용하는 경우, 한 번에 두 개의 시료를 분쇄 할 수있다. 작은 샘플도 표준 연삭 항아리와 볼 밀을 사용하여 처리하기가 어렵습니다. beadbeater을 사용하는 방법 2, 그것의 분쇄 능력에 덜 효과​​적이다. 결과적으로, 보통 CORT 복구는 볼밀 (미공개 데이터)와 비교하여이 방법을 사용하여 약 10 % 낮다. 한편, beadbeater상당히 저렴 볼밀보다, 16-24 번 샘플은 모델에 따라에서 접지 될 수 있고, 상기 방법은 작은 샘플에 매우 적합하다. 때문에 상기 차동 회복, 그것은 특정 연구 내 모든 샘플에 대해 동일한 연삭 방법을 사용하는 것이 바람직하다. 모발 샘플을 처리 한 후, 그들은 원래 타액 CORT을 측정하기위한 민감하고 특정 상용 EIA 키트에 의해 분석된다 추출한 메탄올 CORT로 추출된다. 추출 및 분석 절차는 원숭이의 머리 샘플에서 추출의 시리얼 희석 밀접 본격적인 CORT 표준에서 얻은 측정 값을 병렬 EIA 수치를 산출 시연에 의해 부분적으로 확인되었다. 우리는 그 (플라즈마 및 타액 CORT) 외에도 헤어 CORT 새로운 주택 분기 4,5 원숭이의 관리 상 의무 재배치의 주요 생활 스트레스에 민감한 것으로 나타났다. 프리젠T 종이는 인간과 원숭이의 머리 샘플을 처리하고 이러한 샘플에서 CORT를 추출하고 분석하기 위해 실험실에서 일상적으로 사용되는 방법에 대한 자세한 계정을 제공합니다.

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			<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

스트레스 호르몬 코르티솔 (CORT)은 서서히 인간, 비인간 영장류, 그리고 다른 포유 동물의 성장 머리 샤프트​​에 통합됩니다. 우리는 개발과 검증 된 붉은 털 원숭이의 머리에서 CORT 추출 및 분석하는 방법을 연속적으로 인간의 두피 모발에 사용하기 위해이 방법을 적용. 플라즈마 또는 침에서 얻은 CORT &quot;포인트 샘플&quot;대조적으로, 머리 CORT 호르몬 결합의 기간 동안, 시상 하부 - 뇌하수체 - 부 신피질 (HPA) 시스템 활동의 통합 측정, 따라서 생리적 인 스트레스를 제공합니다. 사람의 두피에 머리가 1cm / 월의 평균 속도로 성장하고 있기 때문에, 머리 부분에서 얻은 CORT 수준의 길이는 몇 cm 잠재적 개월 수에 대한 경험이 스트레스의 바이오 마커 역할을 할 수 있습니다. 우리의 방법에서, 각 모발 샘플은 제 땀과 피지에서 증착 된 머리 샤프트​​의 외부로부터 어떠한 CORT 제거 이소프로판올에 두 번 세척한다. 건조 후,샘플은 모발 단백질의 행렬을 분해 및 추출을위한 표면적을 증가시키는 미세 분말로 분쇄된다. 모간의 내부로부터 CORT 메탄올로 추출하고, 메탄올을 증류 제거하고, 추출액을 분석 완충액에서 재구성된다. 추출 CORT, 표준 및 품질 관리와 함께, 다음 민감하고 특정 시판 효소 면역 측정법 (EIA) 도구에 의해 분석된다. EIA의 판독은 MG 분말 머리 무게 당 PG의 CORT으로 변환됩니다. 이 방법은 인간의 머리 CORT을 분석하기 위해 실험실에서 사용 된, 짧은 꼬리 원숭이, 명주 원숭이, 개, 그리고 북극곰의 몇몇 종. 우리가 실험실에서와 다른 연구 그룹 모두에서 많은 연구가 자연에 만성 스트레스에 노출을 평가뿐만 아니라 실험실 설정에 대한 머리 CORT의 광범위한 적용 가능성을 증명하고있다.

그림 1은 방법 2 분쇄 및 추출을 사용하여 처리 사람의 모발 샘플 (성인 남성과 여성의 인체)의 대표 세트의 출력을 보여줍니다. 컴퓨터 소프트웨어는 데이터 출력을 생성하고 CORT 표준에 4 - 파라미터 S 자형 곡선 (도 2)을 맞추기 위해 사용되었다. 이 판의 사이에 잘​​ CVS는 0.01-5.73 %에서 1.34 %의 평균 내부 분석 CV와 함께였다. 분석 간 CV는 4.41 % 아홉 최근 인간의 머리카락...

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

코르티솔 (CORT)은 인간과 인간이 아닌 영장류의 성장 머리 갱구에 축적. 우리는 높은 정밀도 및 감도로 머리 CORT를 추출하고 분석하는 방법을 설명합니다. 머리 CORT의 측정은 특히 개월 주 기간 동안 만성 스트레스를 평가하기에 적합합니다.

추출 및 인간과 원숭이의 머리에서 코르티솔 분석

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

Research

JoVE Journal

Biology

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

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

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

소일스 및 세디먼츠에서 높은 분자량의 게놈의 DNA의 추출

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

연구

생물학

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.

엘루시브 작은 포유류의 고품질 DNA를 얻기 위해 비침 투 헤어 샘플링 기법

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.

헤어 재생시 상피 - 중간 엽 시그널링의 빠른 유전 분석

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.

에서 프로스타글란딘 추출 및 분석<em&gt; Caenorhabditis의 엘레</em

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.

프로테옴 Transcriptomic 분석에 대한 거미의 독을 추출하고 독 그랜드 Microdissections

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.

그레이 스케일 분석을 사용하여 마우스의 탈모의 정량 분석​​을위한 새로운 기술

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.

히스톤 추출에서 데이터 분석에 : 상향식 질량 분석 사용 히스톤 번역 후 변형 분석을위한 완벽한 워크 플로우

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).

염색질 면역침전 분석을 위한 냉동 키메라 간 조직에서 염색질 추출

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.

인간 골관절염 무릎 관절에서 조직 수집 및 RNA 추출

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.

인간 Antral Follicles에서 계통 특이적 세포의 추출, 표지 및 정제

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