우리는 현장에서 도움 L. 에반스, B. 그레인저, D. Rissling, Z. 심, A. 굿윈, K. 헤이 허스트와 D. 쿠치 감사하고 싶습니다. K. 갈브레스는 친절하게 벨라 쿨라 피카 계곡에서 간 샘플을 제공했습니다. 이 비침 투 유전자 샘플링 방식의 설계에 기여 흥미로운 토론 A. Gonçalves 다 실바와 K. 라센 감사합니다. 이 작품은 자연 과학 및 캐나다의 발견 공학 연구 협의회, 그리고 MAR에 UBC 오카 나간 개별 연구 기금에 의해 투자되었다. 스위스 국립 과학 재단 (National Science Foundation) 박사 원정대 PBSKP3_128523는 PH를 지원. 이 연구는 브리티시 컬럼비아 대학 (A07 - 0126 인증 번호)에서 동물 보호 프로토콜 아래 실시되었다.

1. 헤어 스내어 전에 올무를 설정하는 이상적인 위치는 피카의 서식지 또는 탈루스 슬로프 내에서 결정되어야한다. 이것은 동물이 늦은 여름뿐만 아니라 변소 사이트에서 찾은 신선한 scats에서 수집하는 식물 캐시 아르 헤이 더미가 포함되어 있습니다. 포장 테이프 (길이 10~50cm)의 스트립은 360 ° 끈끈한 표면을 제공하기 위해 올리고 있으며, 봉투 피카의 건초 더미 (그림 1) 또는 변소 사이트의 입구를 웹 같은 방식으로 정렬됩니다. 바위의 구성에 따라 낚싯줄의 조각은 머리 함정의 구조를 지원하는 데 사용하지만,이 출입구의 사용에 대한 자세한 설명 (직경 &lt;30cm) 매우 작은 경우 종종 필요하지 않습니다 수 있습니다 낚싯줄은 헨리와 Russello (2010) 3에서 찾을 수 있습니다. 헤어 스네어스은 가능한 한 자주 확인하고, 머리카락 샘플은 테이프 (그림 2)에 쌓인 거죠 것은 다음 수집하고 표시합니다. 일단 다시 실험실로 이송, 머리카락이 추가 조작까지 살균 집게를 사용하여 끈적끈적한 테이프에서 제거 및 극저온 튜브로 전송하고 -20 ° C에 저장됩니다. 독립적으로 클러스터 샘플이 다른 개인에 속하는 가정하는 동안 머리 올무 함께 모여 헤어 샘플은 하나의 개인에 속하는 것으로 간주됩니다. 후자의 경우에는 머리를 그런 다음 다른 튜브에 배치됩니다. 이러한 가정은 나중에 microsatellite genotypic 데이터에 기반한 DNA 지문과 정체성의 확률의 계산 방법으로 테스트할 수 있습니다. 2. DNA 추출 피카 머리가 매우 얇은하고 작은 루트 전구가 포함되어 있기 때문에, 우리는 이전에 25 머리카락의 최소 양질의 하류 PCR 증폭 3 DNA의 충분한 양을 얻기 위해 필요한 것으로 나타났습니다. 우리는 DNA IQ 시스템 (Promega, 매디슨, WI, 미국)와 머리카락 샘플에서 DNA 격리를 위해 제조 업체의 지침의 약간 수정된 버전을 사용. 첫째, 머리 샘플은 튜브를 여는 동안 머리의 손실을 방지하기 위해 마이크로 원심 분리기에서 다운 소용됩니다. 다음 부화 솔루션은 Proteinase K. 보육의 0.1 M DTT와 1.8ng/ml의 최종 농도 결과 DTT의 10μl (1M) 및 Proteinase K (18ng/ml)의 10μl와 부화 솔루션의 80 μl를 혼합하여 준비 솔루션 (100 μl) 시료에 추가 ° C 1 시간 56 incubated입니다. 그 동안에 용해 용액은 0.01M의 최종 DTT 농도의 결과로, 용해 버퍼의 모든 100 μl에 대한 DTT (1M) 1 μl를 추가하여 준비가되어 있습니다. 준비 용해 용액 (200 μl)는 다음 DNA IQ 수지 7 μl와 함께, 샘플에 추가 고속 3 초 동안 vortexed 5 분 실온에서 incubated입니다. 예제는 다음 2 초 vortexed 및 솔루션에서 자성 비즈의 분리가 발생 마그네틱 스탠드에 삽입됩니다. 나머지 솔루션은 다음 aspirated 및 폐기, 자기 수지의 펠렛을 방해하지 않도록주의하고 있습니다. 예제는 다음 준비 용해 솔루션 vortexed 및 분리가 다시 발생 마그네틱 스탠드에 반환의 100 μl로 처리됩니다. 용해 버퍼 aspirated과 폐기하고,이 단계는 세척 버퍼 (100 μl)를 사용하여 세 번 반복입니다. 예제는 이후 15 분 동안 자기 스탠드에서 건조 남아 있습니다. 일단 건조, 용출 버퍼 100 μl가 샘플에 추가되고 ° C 5 분 65 incubated. 예제는 vortexed 및 자기 스탠드에 삽입됩니다. 지금 eluted DNA를 포함하는 솔루션은 다음 1.5 ML Eppendorf 튜브에 전송하여 추가로 조작 때까지 -20 ° C에 저장됩니다. 간 샘플에서 DNA도 DNA의 IQ 시스템을 사용하여 추출 및 PCR의 amplifications에 대한 긍정적인 제어로 사용되었다. 3. PCR 증폭 우리 비침 투 헤어 올무와 간 샘플에서 얻은 DNA를 그 다음 일반적으로 사용되는 분자 마커 (microsatellite, AFLP, mitochondrial 시토크롬 B와 ZFX / ZFY sexing 표시)의 제품군을 증폭하는 데 사용되었습니다. - 20 NG의 DNA, 각 프라이머의 0.5 μm의, 10 MM 트리스 - HCL (산도 8.3), 50 MM 10 : PCRs가 포함된 25 μL 볼륨 Veriti 열 자전거 타는 사람을 (응용 Biosystems, 포스터 시티, CA, 미국)를 사용하여 수행되었습니다 KCl, 1.5 MM MgCl 2, 200 μm의 dNTPs, 10 μ 소 혈청 알부민 (BSA, 뉴 잉글랜드 Biolabs, 입스 위치, MA, 미국)와 0.5 U AmpliTaq 골드의 DNA 중합 효소 (응용 Biosystems). microsatellite loci를위한 자전거 매개 변수는 터치다운으로 자전거 프로그램 (95시 10 분 사용 최적화되었습니다 ° C, 95 ° 30 S에 대한 C, 30 S는 어닐링 35주기, 72 45의 ° C, 72에서 마지막 단계 다음 ° C 10 분). 1 감소 어닐링 온도 60 ° C에서 55 ° C 사이클 당 29주기 나머지 55에서 계속하는 시점에서 여섯 번째 사이클, ° C.에 도달하기 전까지는 mitochondrial 조각 자전거 매개 변수는 위에서 설명한 것처럼하지만, 소둔 35 사이클로 구성되었습니다50 ° C의 온도 증폭 단편 길이 다형성은 보닌 사에 의해 설명 척추 genomes에 대해 다음과 같은 프로토콜을 실시했다. 마지막으로, 분자 sexing는 HinfI 제한 효소 분해 5 ZFX / ZFY loci의 PCR - RFLP를 사용하여 실시되었다. 소화 ZFX / ZFX 조각이 들어있는 3 % 아가로 오스 겔에 100 BP의 사다리 (뉴잉글랜드 Biolabs)와 함께 실행하는 동안 microsatellite, AFLPs 및 시토크롬 B 시퀀스에서 PCR 제품은, ABI 3130XL 유전자 분석기 (응용 Biosystems)로 실행되었습니다 2.5 %의 SYBR 안전 DNA 젤 얼룩 (Invitrogen, 칼스 배드, CA, 미국)와 RED 개인 젤 이미징 시스템 (알파 Innotech, 샌 리앤드로, CA, 미국)을 사용하여 시각. Microsatellite 및 AFLPs은 Genemapper v3.7 (응용 Biosystems)와 mitochondrial DNA의 크로마토 그램을 사용하면 Sequencher v4.7을 (진 코드 공사 앤 하버, MI, 미국)를 사용하여 시각되었습니다 시각되었습니다. 4. 대표 결과 우리 비침 투 올무를 사용하여 얻은 머리카락 샘플에서 추출한 DNA가 PCR은 분자 마커의 다양한 유형을 증폭하는 데 사용되었습니다. 비교를위한 기초로, DNA가 간 샘플은 우리의 머리카락 샘플과 함께 증폭되었습니다 사용하여 추출. 핵 microsatellite loci의가 성공적으로 머리와 간 샘플 (그림 3) 모두 증폭되었다. 간 샘플을 사용할 때 신호의 강도가 큰했지만,이 유전자형 점수에 부정적인 영향 (그림 3)가 없었어요. AFLPs (그림 4), mitochondrial DNA 시퀀스 (그림 5)와 ZFX / ZFY 분자 sexing 표시 (그림 6)를 사용하면 비슷한 결과가 달성되었습니다. <img src="/files/ftp_upload/2791/2791fig1.jpg" alt="그림 1"/> 그림 1. 비침 투 머리 올무의 예제는 건초 더미에서 설정할 수 있습니다. 올리고 분명히 포장 테이프의 스트립은 건초 더미로 입구를 묶으하는 웹 같은 방식으로 정렬됩니다. 낚싯줄의 조각은 머리 함정에 대한 추가 지원을 제공하기 위해 여기에 사용됩니다. <img src="/files/ftp_upload/2791/2791fig2.jpg" alt="그림 2"/> 그림 2. 패킹 테이프에 붙어있는 머리카락의 다수를 포함하는 성공적인 헤어 올가미. <img src="/files/ftp_upload/2791/2791fig3.jpg" alt="그림 3"/> 그림 3. 대표적인 핵 microsatellite의 크로마토 그램 (Ocp6) 8로 Genemapper v3.7 (응용 Biosystems)에 표시됩니다. 상단 크로마토 그램은 간 조직에서 DNA 증폭에 의해 얻은,이 로커스에서 (358분의 354) heterozygous가되었습니다. 하단 크로마토 그램은 머리카락에서 추출한 DNA를 기반​​으로 heterozygous 유전자형 (362분의 358)를 전시 다른 개인을 나타냅니다. 머리 샘플에서 크로마토 그램의 신호가 간 샘플보다 낮은 강도를 가지고 있지만, 유전자형 점수는 부정적인 신호의 차이에 의해 영향을받지 않습니다. <img src="/files/ftp_upload/2791/2791fig4.jpg" alt="그림 4"/> 그림 4.로서 Genemapper v3.7 (응용 Biosystems)에 표시 대표 AFLP의 크로마토 그램 (E31T32). 상단 크로마토 그램은 간 조직에서 DNA 증폭에 의해 얻은, 하단 크로마토 그램은 머리카락에서 뽑아낸 DNA를 나타냅니다. <img src="/files/ftp_upload/2791/2791fig5.jpg" alt="그림 5"/> 그림 5.로서 Sequencher v4.7 (진 코드 주식 회사)에 표시 mitochondrial DNA 대표 시퀀스 크로마토 그램 (시토크롬 B). 상단 크로마토 그램은 간 조직에서 DNA 증폭에 의해 얻은, 하단 크로마토 그램은 머리카락에서 뽑아낸 DNA를 나타냅니다. <img src="/files/ftp_upload/2791/2791fig6.jpg" alt="그림 6"/> 그림 6. 간 및 헤어 샘플 모두 증폭 대표 분자 sexing 마커 (ZFX / ZFY). 이러한 접근 방식은 Y의 염색체가 X 염색체의가 결여되는이 지역의 HinfI 제한 사이트를 보유하고있는 관측에 의존합니다. 수컷 432 BP, BP 261 및 171 BP의 조각을 제작하는 동안 따라서, 여성은 432 BP의 두 공동 이주 조각을 생산하고 있습니다.

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	<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>

관심 없음 충돌 선언하지 않습니다.

비침 투 유전자 샘플링 여러 가지 이유로 전통적인 라이브 트래핑 방법에 매력적인 대안이되고있다. 첫째, unobtrusively 야생 인구에서 생물 학적 물질 (예, 대변, 머리카락, 깃털, 타액과 점액)​​ 수집하여, 연구자들은 따라서 동물과 연구자 모두에게 위험을 감소, 처리, 심지어 그들을 관찰, 방해없이 인류를 공부할 수 있습니다. 둘째, 비침 투 유전자 샘플링 어려운 희귀 수종의 인구보다 전통적인 라이브 트래핑 방식 6 어려울 수있는 작업을 공부 생물학 자 수 있습니다. 그리고 셋째, NGS 가능성이 있으므로 인구 매개 변수 7 견적에 편견을 최소화하기 위해 도움이, 동물, 샘플링 노력과 비용에 교란을 줄임으로써 샘플 크기를 늘릴 수 있습니다. 위협 수종을 상대할 때는 인구 매개 변수의 편견 견적은 부적 절한 관리가 발생할 수 있으므로이 후자의 요점은, 중요한 증명 수도 있습니다. 현재 동영상 기사에서 우리는 예를 들어 미국의 피카를 사용하여, 어려운 작은 포유류를 샘플에 대한 간단한 소설, 저렴하고 비침 투 방법을 설명합니다. 우리는 머리에서 추출되는 DNA이 비침 투 샘플링 방법 트래핑이나 치명적인 샘플링을 살 수있는 매력적인 대안 만들기, microsatellite, AFLP, mitochondrial 및 sexing 표시에 대하여 간 샘플에서 추출한 DNA와 마찬가지로 수행 보여줍니다. 전반적으로, 우리는 연구에 따라 생물체에 미치는 영향을 최소화하면서이 방법은 희귀하거나 어려운 작은 포유류 종의 유전자 인구 및 행동 연구의 데이터 수집 단계에 유용하게 사용될 것으로 예상.

헤어 스내어 : <ul><li> 맑은 포장 테이프 롤 (3M, 세인트 폴, 미네소타, 미국) </li><li> 낚싯줄 (10파운드) </li><li> 극저온 튜브 (2ml) </li><li> 무균 포셉 </li><li> 액체 질소 듀어 (선택 사항) </li></ul> DNA 추출 : <ul><li> 조직 및 헤어 추출 키트 (; Promega Cat. # DC6740)로, DNA IQ 시스템 (Promega Cat. # DC6701). </li><li> MagneSphere 기술 자기 분리 스탠드 (Cat. # Z5342, Promega) </li><li> 마이크로 원심 분리기 </li><li> 하이브 리다이 제이션 오븐이나 물 목욕 </li><li> 1.5 ML Eppendorf 튜브 </li></ul>

a noninvasive hair sampling technique to obtain high quality dna from elusive small mammals

비침 투 유전자 샘플링 방식은 야생 동물의 인구를 공부하는 것이 점차 중요 해지고 있습니다. 연구의 숫자는 인구 유전 야생 인구 1 인구 통계학 조사를 비침 투 샘플링 기법을 사용했다고합니다. 이러한 접근 방식은 희귀하거나 어려운 수종 2 거래할 때 특히 유용하게 입증되었습니다. 이러한 방법의 숫자가 샘플 머리, 대변과 육식 동물과 중간 크기의 포유류에서 다른 생물 소재 개발되었습니다 동안, 그들은 크게 어려운 작은 포유류에 안된 남아있다. 이 비디오에서는, 우리는 소설, 어려운 작은 포유류, 미국 피카 (Ochotona princeps)를 대상으로 저렴한 가격과 비침 투 헤어 올무를 제시한다. 우리는 설정 테이프가 웹과 같은 방식으로 배열하고 pikas &#39;서식지의 경로를 따라 여행을 배치 포장의 스트립으로 구성되어 머리 함정의 일반적인 설명합니다. 그러면 수집하고 연구실로 돌아온 수있는 머리의 대량 수집에있는 함정의 효율성을 보여줍니다. 그러면 DNA를 분리하고 핵 microsatellites, 증폭된 파편 길이 polymorphisms (AFLPs), mitochondrial 시퀀스 (800bp)뿐만 아니라 포함하여 일반적으로 사용되는 분자 마커를 증폭하기 위해이 방법의 유틸리티를 감상할 수있는 DNA IQ 시스템 (Promega)의 사용을 보여줍니다 분자 sexing 표시. 전반적으로, 우리는 야생 동물의 인구 생물학을위한 샘플링 기법으로이 소설 비침 투 헤어 올무의 유틸리티를 보여줍니다. 우리는이 접근 방식이 연구 생물에 미치는 영향을 최소화하면서, 자연 인구 이내에 조사의 영역을 개방, 작은 포유류의 다양한 적용됩니다 것으로 예상.

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

저희는 미국 피카에 표시 효율적으로 어려운 작은 포유류에서 머리카락 샘플을 수집하는 비침 투 샘플링 방법을 제시한다. 우리는 샘플 머리카락에서 DNA를 추출하여 일반적으로 야생 동물의 생태와 보존 연구에 사용된 분자 마커의 여러 유형을 확장하여이 방법의 유틸리티를 보여줍니다.

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 ...

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Biology

20.7K 조회수.  University of British Columbia, Okanagan Campus. 저희는 미국 피카에 표시 효율적으로 어려운 작은 포유류에서 머리카락 샘플을 수집하는 비침 투 샘플링 방법을 제시한다. 우리는 샘플 머리카락에서 DNA를 추출하여 일반적으로 야생 동물의 생태와 보존 연구에 사용된 분자 마커의 여러 유형을 확장하여이 방법의 유틸리티를 보여줍니다.

비디오: 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.

인간의 사후 뇌 조직에서 단세포 집단의 고품질 RNA 구하기

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

연구

생물학

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.

비침 투 VIVO에서 스몰 애니멀 MRI 및 MRS : 기본 실험 절차

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

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

히타치 F - 7000 형광 분광 및 PicoGreen 염료를 사용하여 dsDNA의 부량

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

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.

의 DNA Microarrays : 샘플 품질 제어, 배열 하이브 리다이 제이션 및 스캔

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.

미생물 매트에서 높은 분자량의 DNA의 추출

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

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

파우더 샘플의 절대 양자 수율 측정

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

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.

높은 처리량 시퀀싱을위한 고품질 염색질 면역 침전의 DNA 템플릿의 생성 (칩 SEQ)

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.

Zebrafish의 배아를 사용하여 높은 처리량 접근 수동 작은 분자 화면

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.

가금류 생산 샘플에서 세균 공동체의 정성 및 정량 평가를위한 하이브리드 DNA 추출 방법

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 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.