MT의 연구실에서 연구는 국립 과학 재단 (DBI - 0820610 및 IOS - 1022102)와 건강의 뉴욕과 (NYSTEM - C024308)에서 기금에 의해 지원됩니다. CM은 교육과 과학 (2007-0937)와 재단 라파엘 델 피노, 스페인의 스페인 교육부에서 박사 장학금을 받았습니다.

<ol>
	<li>Rensink, W. A., Buell, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+expression+profiling+resources+for+plant+genomics.">Microarray expression profiling resources for plant genomics.</a> Trends. Plant. Sci. 10, 603-609 (2005).</li><li>Li, P., Ponnala, L., Gandotra, N., Wang, L., Si, Y., Tausta, S. L., Kebrom, T. H., Provart, N., Patel, R., Myers, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+dynamics+of+the+maize+leaf+transcriptome.">The developmental dynamics of the maize leaf transcriptome.</a> Nat. Genet. 42, 1060-1067 (2010).</li><li>Lister, R., O'Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, A. H., Ecker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+integrated+single-base+resolution+maps+of+the+epigenome+in+Arabidopsis.">Highly integrated single-base resolution maps of the epigenome in Arabidopsis.</a> Cell. 133, 523-536 (2008).</li><li>Brady, S. M., Orlando, D. A., Lee, J. Y., Wang, J. Y., Koch, J., Dinneny, J. R., Mace, D., Ohler, U., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-resolution+root+spatiotemporal+map+reveals+dominant+expression+patterns.">A high-resolution root spatiotemporal map reveals dominant expression patterns.</a> Science. 318, 801-806 (2007).</li><li>Brooks, L., Strable, J., Zhang, X., Ohtsu, K., Zhou, R., Sarkar, A., Hargreaves, S., Elshire, R. J., Eudy, D., Pawlowska, T., Ware, D., Janick-Buckner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdissection+of+shoot+meristem+functional+domains.">Microdissection of shoot meristem functional domains.</a> PLoS. Genet. 5, e1000476-e1000476 (2009).</li><li>Galbraith, D. W., Birnbaum, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+studies+of+cell+type-specific+gene+expression+in+plants.">Global studies of cell type-specific gene expression in plants.</a> Annu. Rev. Plant. Biol. 57, 451-475 (2006).</li><li>Ohtsu, K., Smith, M., Emrich, S., Borsuk, L., Zhou, R., Chen, T., Zhang, X., Timmermans, M., Beck, J., Buckner, B., Janick-Buckner, D., Nettleton, D., Scanlon, M., Schnable, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+analysis+of+the+shoot+apical+meristem+of+maize+(Zea+mays+L.).">Global gene expression analysis of the shoot apical meristem of maize (Zea mays L.).</a> Plant. J. 52, 391-404 (2007).</li><li>Juarez, M. T., Kui, J. S., Thomas, J., Heller, B. A., Timmermans, M. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA-mediated+repression+of+rolled+leaf1+specifies+maize+leaf+polarity.">microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity.</a> Nature. 428, 84-88 (2004).</li><li>Kramer, E. M., Irish, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+genetic+mechanisms+controlling+petal+development.">Evolution of genetic mechanisms controlling petal development.</a> Nature. 399, 144-148 (1999).</li><li>Stuurman, J., Jäggi, F., Kuhlemeier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shoot+meristem+maintenance+is+controlled+by+a+GRAS-gene+mediated+signal+from+differentiating+cells.">Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells.</a> Genes Dev. 16, 2213-2218 (2002).</li><li>Kim, M., McCormick, S., Timmermans, M., Sinha, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+domain+of+PHANTASTICA+determines+leaflet+placement+in+compound+leaves.">The expression domain of PHANTASTICA determines leaflet placement in compound leaves.</a> Nature. 424, 438-443 (2003).</li><li>Kramer, E. M., Holappa, L., Gould, B., Jaramillo, M. A., Setnikov, D., Santiago, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elaboration+of+B+gene+function+to+include+the+identity+of+novel+floral+organs+in+the+lower+eudicot+Aquilegia.">Elaboration of B gene function to include the identity of novel floral organs in the lower eudicot Aquilegia.</a> Plant Cell. 19, 750-766 (2007).</li><li>Lippman, Z. B., Cohen, O., Alvarez, J. P., Abu-Abied, M., Pekker, I., Paran, I., Eshed, Y., Zamir, 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+making+of+a+compound+inflorescence+in+tomato+and+related+nightshades.">The making of a compound inflorescence in tomato and related nightshades.</a> PLoS Biol. 6, e288-e288 (2008).</li><li>Kidner, C., Timmermans, M. C. 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+hybridization+as+a+tool+to+study+the+role+of+microRNAs+in+plant+development.">In situ hybridization as a tool to study the role of microRNAs in plant development.</a> Methods. Mol. Biol. 342, 159-179 (2006).</li><li>Jackson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+labeling+of+KNOTTED1+mRNA+and+protein+reveals+multiple+potential+sites+of+protein+trafficking+in+the+shoot.">Double labeling of KNOTTED1 mRNA and protein reveals multiple potential sites of protein trafficking in the shoot.</a> 129, 1423-1429 (2002).</li><li>Jensen, W. 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> Botanical Histochemistry. , 408-408 (1962).</li><li>Jackson, D., Bowles, D. J., Gurr, S. J., McPherson, M. <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+hybridization+in+plants.">In situ hybridization in plants.</a> Molecular Plant Pathology: A Practical Approach. , 163-174 (1991).</li><li>Chitwood, D. H., Nogueira, F. T., Howell, M. o. n. t. g. o. m. e. r. y., Carrington, T. A., C, J., Timmermans, M. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+via+small+RNA+mobility.">Pattern formation via small RNA mobility.</a> Genes. Dev. 23, 549-554 (2009).</li><li>Piette, D., Hendrickx, M., Willems, E., Kemp, C. R., Leyns, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+procedure+for+whole-mount+in+situ+hybridization+on+mouse+embryos+and+embryoid+bodies.">An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies.</a> Nat. Protoc. 3, 1194-1201 (2008).</li><li>Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R., Aida, M., Palme, K., Scheres, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PIN+auxin+efflux+facilitator+network+controls+growth+and+patterning+in+Arabidopsis+roots.">The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots.</a> Nature. 433, 39-44 (2005).</li><li>Jackson, D., Hake, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+phyllotaxy+in+maize+by+the+abphyl1+gene.">Control of phyllotaxy in maize by the abphyl1 gene.</a> Development. , 126-315 (1999).</li><li>Chuck, G., Whipple, C., Jackson, D., Hake, 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+maize+SBP-box+transcription+factor+encoded+by+tasselsheath4+regulates+bract+development+and+the+establishment+of+meristem+boundaries.">The maize SBP-box transcription factor encoded by tasselsheath4 regulates bract development and the establishment of meristem boundaries.</a> Development. 137, 1243-1250 (2010).</li><li>Long, J. A., Barton, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+apical+embryonic+pattern+in+Arabidopsis.">The development of apical embryonic pattern in Arabidopsis.</a> Development. 125, 3027-3035 (1998).</li><li>Nogueira, F. T., Chitwood, D. H., Madi, S., Ohtsu, K., Schnable, P. S., Scanlon, M. J., Timmermans, M. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+small+RNA+accumulation+in+the+maize+shoot+apex.">Regulation of small RNA accumulation in the maize shoot apex.</a> PLoS Genet. 5, e1000320-e1000320 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기에 명시된 현장 하이브 리다이 제이션 방식의 좋은 세포 해상도, 높은 감도와 특이성과 관심의 유전자의 spatiotemporal 표현 패턴의 직접 시각화 수 있습니다. 프로토콜은 유전자 간의 표현 수준의 양적 비교를 허용하지 않습니다. 그러나, 방법의 높은 감도와 해상도가 유전자 발현을 분석 대부분의 다른 방법으로 가능하지 않은 조직이 8, 18, ​​내의 유전자 발현의 그라디언트를 공개하실 수 있습니다. 프로토콜 문제 해결은 문제가 만들 수있는 단계가 포함되어 있습니다. 프로토콜의 가장 중요한 단계는 조직의 고정 및 프로브의 선택과 라벨입니다. 낮은 발굴의 설립과 함께 저조한 침투 조직 및 프로브는 배경 위에 감지하기 어려울 수도 매우 약한 신호를 얻을 것입니다. 관심 각각의 새로운 유전자 들어, diffe에서 파생된 몇 가지 뚜렷한 프로브를 시도하는 것이 좋습니다명시 지역을 임대. 때때로, 유전자의 다른 작은 지역을 대상으로 두 개 또는 세 탐사선이 강하고 특정 하이브 리다이 제이션 신호를 얻기 위해 결합해야 할 수도 있습니다. 또한, 같은 온도와 하이브 리다이 제이션 버퍼 구성으로 하이브 리다이 제이션 조건, 각각의 유전자 또는 하이브 리다이 제이션 엄중을 낮추거나 높일 조직에 대해 최적화할 수 있습니다. 또한 단백 분해 효소 처리 단계는 변수이며 다른 식물 종 (种) 또는 매우 낮은 풍부한 성적의 검출을위한 프로토콜을 적응 변경할 수 있습니다. 모두 긍정적이고 부정적인 제어 프로브의 포함은 프로토콜의 문제 지점을 식별하는데 도움이 될 것입니다. 절차는 파라핀 - 임베디드 식물 조직 섹션에 최적화되어 있지만 사소한 수정은 현장 하이브리드화에서 전체 마운트에도 사용할 수 있습니다로. 널리 원위치 하이브리드화에서 동물 연구 19 전체 마운트에 사용되는 것은 있지만 리터 것입니다이러한 배아, 뿌리 또는 젊은 meristematic 조직 20,21 쉽게 침투 수있는 식물 조직에 imited. 프로토콜은 쉽게 동시에 두 개의 mRNAs를 검색하거나 단백질 15,22,23 함께 성적표를 집중하기 위해 수정할 수 있습니다. 마지막으로, 그것은 식물의 작은 RNA 표현 패턴의 시각화에이 방법의 응용 프로그램을 확장하는 것이 가능합니다. miRNA 발현 패턴은 옥수수와 Arabidopsis 8,14 모두에서이 프로토콜을 사용하여 결정되었습니다. 최근 체외 베꼈는데 프로브에있는가에 의해 대체되었습니다 상용 파다 - 레이블 잠금 핵산 (LNA) oligo 프로브 그 증가 신호 감도 18, 24.

<table border="1"><tbody><tr><th> 제품 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 비고 </th></tr><tr><td> Cytoseal 60 4온스 </td><td> 어부 </td><td> 23-244-256 </td><td> 톨루엔 기반 8310-4 </td></tr><tr><td> Histoclear </td><td> 어부 </td><td> 50-899-90147 </td><td></td></tr><tr><td> 조직 경로 Paraplast 플러스 </td><td> 어부 </td><td> 23-021-400 </td><td></td></tr><tr><td> N + 나일론 막 Hybond - N + </td><td> GE 헬스케어 </td><td> RPN203B </td><td></td></tr><tr><td> RNase OUT, 40 U / μL </td><td> Invitrogen </td><td> 10777019 </td><td></td></tr><tr><td> RQ1 RNase - 무료 DNase, 1 U / μl </td><td> Promega </td><td> M6101 </td><td></td></tr><tr><td> 발굴 - 라벨 컨트롤 RNA </td><td> 호슈 </td><td> 11585746910 </td><td></td></tr><tr><td> 발굴 RNA 라벨 믹스 </td><td> 호슈 </td><td> 11277073910 </td><td></td></tr><tr><td> SP6 RNA 효소 1,000 U </td><td> 호슈 </td><td> 10810274001 </td><tD&gt; </td></tr><tr><td> T3 RNA 효소 1,000 U </td><td> 호슈 </td><td> 11031163001 </td><td></td></tr><tr><td> T7 RNA 효소 1,000 U </td><td> 호슈 </td><td> 10881767001 </td><td></td></tr><tr><td> RNase - A </td><td> 호슈 </td><td> 109,142 </td><td> 글리세롤 50% 트리스 100mM pH8.0에서 용해 </td></tr><tr><td> 시약을 차단 </td><td> 호슈 </td><td> 1 096 176 </td><td> ° C TBS 버퍼 70 개까지 열 </td></tr><tr><td> 안티 Digoxygenin - AP, 양 150 U의 팹 조각 </td><td> 호슈 </td><td> 1 093 274 </td><td></td></tr><tr><td> NBT / BCIP 재고 솔루션 </td><td> 호슈 </td><td> 1 681 451 </td><td></td></tr><tr><td> 미니 빠른 스핀 RNA 열 </td><td> 호슈 </td><td> 11814427001 </td><td></td></tr><tr><td> E.에서 tRNA 대장균 </td><td> 호슈 </td><td> 109,541 </td><td></td></tr><tr><td> 트라 이튼 ® X - 100 </td><td> 시그마 </td><td> T8787 </td><td></td></tr><tr><td> 십대 초반 - 2​​0</td><td> 시그마 </td><td> P9416 </td><td></td></tr><tr><td> Deionized 포름 아미드 </td><td> 시그마 </td><td> F9037 </td><td></td></tr><tr><td> Streptomyces griseus 유형 XIV에서 프로 테아제 </td><td> 시그마 </td><td> P5147 </td><td> 37 milliQ 물, 사전 다이제스트 4 시간 만에 희석 ° C </td></tr><tr><td> Triethanolamine </td><td> 시그마 </td><td> 90,279 </td><td> 물에 희석, HCL와 pH8.0 데리고 </td></tr><tr><td> 아세트산 무수물 </td><td> 시그마 </td><td> A6404 </td><td></td></tr><tr><td> Leuconostoc 종에서 Dextran 황산 나트륨 소금. </td><td> 시그마 </td><td> D8906 - 5G </td><td> Dextran 황산은 ° C가 용해에 80 가열한다. </td></tr><tr><td> Denhardt의 솔루션 50x 컨센트레이트 </td><td> 시그마 </td><td> D2532 </td><td></td></tr><tr><td> 소 혈청 알부민에서 - ≥ 98% </td><td> 시그마 </td><td> A7906 </td><td></td></tr><tr><td> Eosin Y 나트륨 소금 </td><td> 시그마 </td><td> E4382 </td><td> 포화까지 96~100%의 에탄올에 용해 </td></tr><tr><td> Paraformaldehyde </td><td> 시그마 </td><td> P6148 </td><td></td></tr><tr><td> 에탄올 절대 200 증명 </td><td></td><td></td><td></td></tr><tr><td> 페놀 / 클로로포름 </td><td></td><td></td><td></td></tr><tr><td> 기본 틀 </td><td> 전자 Microsocpy 과학 </td><td> 62352-15 </td><td></td></tr><tr><td> 퍼가기 반지 </td><td> 어부 </td><td> 22-038-197 </td><td></td></tr><tr><td> 모자와 20ml 일회용 섬광 튜브, </td><td> VWR </td><td> 66020-326 </td><td></td></tr><tr><td> 프로브 -에서 - 플러스 슬라이드 </td><td> 어부 </td><td> 22-230-900 </td><td></td></tr><tr><td> 마이크로 커버 24x50 mm 안경 </td><td> VWR </td><td> 48404-452 </td><td></td></tr><tr><td> 워트 먼지 용지 3mm </td><td> VWR </td><td> 89022-360 </td><td></td></tr><tr><td> 세 유리 요리 한 - 안녕 -nless 철강 슬라이드 선반 </td><td> 전자 Microsocpy 과학 </td><td> 71420-25 </td><td> 두 유리 histoclear 치료에 필요한 걸려, 마지막 하나는 아세트산 무수물 치료에 사용됩니다 </td></tr><tr><td> 18 깨끗한 플라스틱 용기 </td><td> 전자 Microsocpy 과학 </td><td> 71,402 </td><td></td></tr><tr><td> sea​​lable 뚜껑 2 개 또는 3 크고 평평한 플라스틱 상자 </td><td></td><td></td><td> 하이브 리다이 제이션 및 항체 단계에 필요한 </td></tr><tr><td> 좋은 브러쉬 </td><td></td><td></td><td> 왁스 리본을 나눠 도움말 </td></tr><tr><td> 마이크로톰 </td><td></td><td></td><td> Leica </td></tr><tr><td> 셀프 클리닝 드라이 진공 시스템 </td><td> 웨일스 사람 </td><td> 2025 </td><td></td></tr><tr><td> 따뜻한 슬라이드 </td><td> 어부 </td><td></td><td></td></tr><tr><td> 가열 블록 </td><td></td><td></td><td> 60 ° C와 85 ° C에서 필요</td></tr><tr><td> 58-60시 인큐베이터 ° C </td><td> 어부 </td><td></td><td> 용융 Paraplast과 단계 </td></tr><tr><td> 55 ° C와 37 ° C에 대한 오븐이나 물을 욕조 </td><td> 어부 </td><td></td><td> 온도 사이의 빠른 변화로 인해 두 필요 </td></tr><tr><td> 원심 분리기 (4 ° C - 20 ° C) </td><td></td><td></td><td></td></tr><tr><td> 퓸 후드 </td><td></td><td></td><td></td></tr></tbody></table>

in situ hybridization for the precise localization of transcripts in plants

몇 십년의 게놈 연구의 발전과 함께, 식물 생물학은 유전자 발현의 대규모 양적 분석을 제시 수많은 연구를 볼 수 있습니다. Microarray 및 차세대 시퀀싱 접근 방식은, 발달, 생리학 및 스트레스 반응 과정을 조사 epigenetic과 작은 RNA 경로를 해부하다, 그리고 1-3 대규모 유전자 규제 네트워크를 구축하는 데 사용되고있다. 이 기술이 대규모 유전자 세트의 동시 분석을 용이하게하는 동안, 그들은 일반적으로 유전자 발현 변화의 매우 제한된 spatiotemporal 해상도를 제공합니다. 이 제한은 부분적으로 lasermicrodissection 또는 4-7를 정렬 형광 - 활성 세포와 함께 방법을 사용하거나 프로 파일링하여 극복할 수 있습니다. 그러나, 완전히 유전자의 생물 학적 역할을 이해하기 위해, 세포 해상도로 표현의 spatiotemporal 패턴에 대한 지식이 필수적입니다. 특히, 개발 또는 환경 STI의 효과를 공부하면muli 및 돌연변이 유전자의 표현 패턴의 상세한 분석이 필수적이 될 수 있습니다. 특정 세포 유형에 표현의 손실 또는 이득과 관련된 경우 예를 들어, 핵심 규제 유전자의 표현 수준에 미묘한 양적 차이가 극적인 phenotypes 발생할 수 있습니다. 여러 가지 방법은 정기적으로 유전자 발현 패턴의 세부 심사에 사용됩니다. 하나는 유전자 변형 기자 라인의 분석을 통해이다. 여러 개의 유전자를 분석하거나 변화에 완강히 저항하는 식물에서 작업할 때 이러한 분석은, 그러나, 시간이 많이 걸리는 될 수 있습니다. 또한, transgene 표현식 패턴이 내생 유전자의를 모방 수 있도록 독립적인 검증은 일반적으로 필요합니다. 현장 하이브 리다이 제이션에 Immunohistochemical 단백질 지방화 또는 mRNA는 세포와 조직 내에서 유전자 발현의 직접적인 시각화에 대한 비교적 빠른 대안을 제시한다. 후자는 쉽게 U 수있는 독특한 장점을 가지고관심의 유전자에 SED. 원위치 하이브 리다이 제이션에 관심의 유전자의 시험 관내 전사에 의해 얻은 프로브 표시 안티 - 감각의 RNA와 하이브리드화하여 세포 표적 mRNAs의 탐지 수 있습니다. 여기 우리는 높은 감도와 구체적인 식물의 유전자 표현의 현장 지방화에 대한 프로토콜을 설명합니다. 그것은 paraformaldehyde에 고정, 파라핀 - 임베디드 섹션, 조직학 우수 보존을주고 단백질 감지 및 알칼리성 인산 가수 분해 효소 - colorimetric 반응에 의해 시각 아르 발굴 - 분류 프로브와 함께 사용하기위한 최적화되었습니다. 이 프로토콜은 성공적으로 식물 종의 다양한 조직의 숫자에 적용되었으며, mRNAs뿐만 아니라 작은 RNAs 8-14의 표현을 분석하는 데 사용할 수 있습니다.

Watch this Scientific Journal Video about In Situ Hybridization for the Precise Localization of Transcripts in Plants at JoVE.com

In Situ Hybridization for the Precise Localization of Transcripts in Plants

<em&gt; 현장에서</em여기에 설명된&gt; 하이브 리다이 제이션 프로토콜은 높은 민감도와 특이성과 세포 수준에서 mRNA 작은 RNA 표현의 직접적인 지역화 가능합니다. 절차는, 파라핀 - 임베디드 식물 조직 섹션에 최적화된 식물과 조직의 다양한 적용, 10 일 이내에 완료될 수 있습니다.

식물의 성적 증명서의 정확한 현지화에 대한 원위치 하이브리드화에서

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

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In Situ Hybridization for the Precise Localization of Transcripts in Plants

51.1K 조회수.  Cold Spring Harbor Laboratory.  현장에서 하이브 리다이 제이션 프로토콜은 높은 민감도와 특이성과 세포 수준에서 mRNA 작은 RNA 표현의 직접적인 지역화 가능합니다. 절차는, 파라핀 - 임베디드 식물 조직 섹션에 최적화된 식물과 조직의 다양한 적용, 10 일 이내에 완료될 수 있습니다.

비디오: In Situ Hybridization for the Precise Localization of Transcripts in Plants

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

조기 칙 태아의 현장 하이브리드화에서 더블 전체 산

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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생물학

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

형광 현장에서</em인간의 정자에서> 하이브리드화 (물고기) 프로토콜

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

방사성 현장에서</em조직에서 다양한 유전자 발현 패턴을 검출> 하이브리드화

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

형광에 의한 바이러스성 RNA의 탐지<em&gt; 현장에서</em&gt; 하이브리드화 (물고기)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

형광등과 결합 염색체 복제 타이밍<em&gt; 현장에서</em&gt; 하이브리드

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

전체 마운트 RNA 형광<em&gt; 현장에서</em의&gt; 하이브리드<em&gt; Drosophila</em&gt; 태아

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

마이크로 RNA<em&gt; 현장에서</em&gt; 포르말린 고정 신장 조직에 대한 하이브리드

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

형광 DNA를위한 간단한 방법<em&gt; 현장에서</em&gt; 숙청 염색체에 하이브리드

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.