Pesquisa no laboratório de MT é suportado por concessões do National Science Foundation (DBI-0820610-1022102 e IOS) e do Departamento de NY da Saúde (NYSTEM-C024308). CM recebeu bolsas de pós-doutorado do Ministério espanhol da Educação e Ciência (2007-0937) e da Fundação Rafael del Pino, Espanha.

<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>

Não há conflitos de interesse declarados.

O método de hibridização in situ descrito aqui permite a visualização direta do padrão de expressão espaço-temporal de qualquer gene de interesse, com resolução de celular grande, de alta sensibilidade e especificidade. O protocolo não permite uma comparação quantitativa dos níveis de expressão entre os genes. No entanto, de alta sensibilidade do método e da resolução pode revelar gradientes de expressão de genes dentro de um tecido de 8, 18, ​​que não é possível com a maioria dos outros métodos de análise de expressão gênica. O protocolo inclui várias etapas, que podem fazer de resolução de problemas problemático. Os passos mais críticos do protocolo são a fixação do tecido e da seleção e rotulagem da sonda. Tecidos mal infiltrado e sondas com incorporação DIG baixa renderá sinais muito fracos que podem ser difíceis de detectar acima de fundo. Para cada novo gene de interesse, é aconselhável tentar algumas sondas distintas derivadas de difealugar regiões da transcrição. Ocasionalmente, duas ou três sondas de segmentação diferentes regiões menores do gene pode precisar ser combinados para obter um sinal forte e hibridização específica. Além disso, as condições de hibridização, como a temperatura ea composição do tampão de hibridação, pode ser otimizado para cada gene ou tecido para diminuir ou aumentar o rigor de hibridização. Também a etapa de tratamento protease é uma variável e pode ser alterado para adaptar o protocolo para diferentes espécies de plantas ou para a detecção de transcritos com abundância muito baixa. A inclusão de ambas as sondas de controlo positivo e negativo será útil na identificação de pontos problemáticos do protocolo. O procedimento é otimizado para parafina cortes de tecidos vegetais, mas com pequenas modificações podem ser usados ​​também para todo o monte de hibridização in situ. Embora amplamente utilizados em animais de pesquisa 19, montar toda a hibridização in situ será limited de tecidos vegetais que podem ser facilmente infiltrado, como embriões, raízes ou jovens tecidos meristemáticos 20,21. O protocolo também pode ser facilmente modificado para detectar simultaneamente dois mRNAs distintos ou localizar transcrições junto proteínas 15,22,23. Finalmente, é possível estender a aplicação deste método para a visualização de pequenas padrões de expressão do RNA em plantas. padrões de expressão de miRNA foram determinados utilizando este protocolo em ambos milho e Arabidopsis 8,14. Mais recentemente, o em sondas transcritos in vitro foram substituídos por comercialmente disponíveis DIG marcado bloqueado ácidos nucleicos (LNA) sondas oligo que aumentam a sensibilidade do sinal 18, 24.

<table border="1"><tbody><tr><th> Produto </th><th> Companhia </th><th> Número de catálogo </th><th> Observações </th></tr><tr><td> Cytoseal 60 4 oz </td><td> Pescador </td><td> 23-244-256 </td><td> Tolueno baseado em 8310-4 </td></tr><tr><td> Histoclear </td><td> Pescador </td><td> 50-899-90147 </td><td></td></tr><tr><td> Além disso tecido Path Paraplast </td><td> Pescador </td><td> 23-021-400 </td><td></td></tr><tr><td> N + membrana de nylon Hybond-N + </td><td> GE Healthcare </td><td> RPN203B </td><td></td></tr><tr><td> RNase OUT; 40 U / mL </td><td> Invitrogen </td><td> 10777019 </td><td></td></tr><tr><td> RQ1 RNase-Free DNase; 1 U / mL </td><td> Promega </td><td> M6101 </td><td></td></tr><tr><td> DIG marcado Controle RNA </td><td> Roche </td><td> 11585746910 </td><td></td></tr><tr><td> DIG RNA mix rotulagem </td><td> Roche </td><td> 11277073910 </td><td></td></tr><tr><td> SP6 polimerase RNA 1.000 U </td><td> Roche </td><td> 10810274001 </td><td&gt; </td></tr><tr><td> RNA polimerase T3 1.000 U </td><td> Roche </td><td> 11031163001 </td><td></td></tr><tr><td> T7 RNA polimerase 1000 U </td><td> Roche </td><td> 10881767001 </td><td></td></tr><tr><td> RNase-A </td><td> Roche </td><td> 109142 </td><td> dissolver em glicerol 50% pH8.0 Tris 100mM </td></tr><tr><td> Bloqueio de reagente </td><td> Roche </td><td> 1 096 176 </td><td> Calor de até 70 ° C em tampão TBS </td></tr><tr><td> Anti-digoxigenina-AP, fragmentos Fab de ovelha 150 U </td><td> Roche </td><td> 1 093 274 </td><td></td></tr><tr><td> NBT / BCIP solução estoque </td><td> Roche </td><td> 1 681 451 </td><td></td></tr><tr><td> mini-rotação rápida Colunas RNA </td><td> Roche </td><td> 11814427001 </td><td></td></tr><tr><td> tRNA de E. coli </td><td> Roche </td><td> 109541 </td><td></td></tr><tr><td> Triton ® X-100 </td><td> Sigma </td><td> T8787 </td><td></td></tr><tr><td> Tween-20</td><td> Sigma </td><td> P9416 </td><td></td></tr><tr><td> Formamida deionizada </td><td> Sigma </td><td> F9037 </td><td></td></tr><tr><td> Protease de Streptomyces griseus Tipo XIV </td><td> Sigma </td><td> P5147 </td><td> Diluir em água MilliQ e pré-digerir 4 horas a 37 ° C </td></tr><tr><td> Trietanolamina </td><td> Sigma </td><td> 90279 </td><td> Diluída em água, leve ao pH8.0 com HCl </td></tr><tr><td> Anidrido acético </td><td> Sigma </td><td> A6404 </td><td></td></tr><tr><td> Dextran sal sulfato de sódio a partir de Leuconostoc spp. </td><td> Sigma </td><td> D8906-5G </td><td> Sulfato de Dextran deve ser aquecido a 80 ° C para dissolver. </td></tr><tr><td> Solução Denhardt de Concentrado de 50x </td><td> Sigma </td><td> D2532 </td><td></td></tr><tr><td> Albumina de soro bovino - ≥ 98% </td><td> Sigma </td><td> A7906 </td><td></td></tr><tr><td> Eosina Y sal dissódico </td><td> Sigma </td><td> E4382 </td><td> Dissolver em etanol 96-100% até saturada </td></tr><tr><td> Paraformaldeído </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> Etanol absoluto prova 200 </td><td></td><td></td><td></td></tr><tr><td> Fenol / clorofórmio </td><td></td><td></td><td></td></tr><tr><td> Base de Dados de moldes </td><td> Eletrônicos Microsocpy Ciência </td><td> 62352-15 </td><td></td></tr><tr><td> Anéis de incorporação </td><td> Pescador </td><td> 22-038-197 </td><td></td></tr><tr><td> 20ml descartáveis ​​frascos de cintilação, com tampas </td><td> VWR </td><td> 66020-326 </td><td></td></tr><tr><td> Sonda-on-plus slides </td><td> Pescador </td><td> 22-230-900 </td><td></td></tr><tr><td> Óculos de cobrir Micro 24x50 milímetros </td><td> VWR </td><td> 48404-452 </td><td></td></tr><tr><td> Whatman 3 milímetros de papel </td><td> VWR </td><td> 89022-360 </td><td></td></tr><tr><td> Três pratos de vidro e um staiporta-lâminas de aço NLES </td><td> Eletrônicos Microsocpy Ciência </td><td> 71420-25 </td><td> Duas de vidro côncavo são necessárias para o tratamento histoclear eo último é utilizado para o tratamento de anidrido acético </td></tr><tr><td> 18 recipientes de plástico limpos </td><td> Eletrônicos Microsocpy Ciência </td><td> 71402 </td><td></td></tr><tr><td> 2 ou 3 grandes plana caixas de plástico com tampa selável </td><td></td><td></td><td> necessários para as etapas de hibridização e de anticorpos </td></tr><tr><td> Pincéis finos </td><td></td><td></td><td> Ajuda distribuindo fitas de cera </td></tr><tr><td> Micrótomo </td><td></td><td></td><td> Leica </td></tr><tr><td> Auto-limpeza a seco sistema de vácuo </td><td> Ludibriar </td><td> 2025 </td><td></td></tr><tr><td> Deslize mais quentes </td><td> Pescador </td><td></td><td></td></tr><tr><td> Bloco de aquecimento </td><td></td><td></td><td> necessários a 60 ° C e 85 ° C</td></tr><tr><td> Incubadora a 58-60 ° C </td><td> Pescador </td><td></td><td> Para as etapas com Paraplast fundido </td></tr><tr><td> Fornos ou banhos de água de 55 ° C e 37 ° C </td><td> Pescador </td><td></td><td> Precisa de dois, devido à mudança rápida entre as temperaturas </td></tr><tr><td> Centrifugação (4 ° C - 20 ° C) </td><td></td><td></td><td></td></tr><tr><td> Coifa </td><td></td><td></td><td></td></tr></tbody></table>

in situ hybridization for the precise localization of transcripts in plants

Com os avanços na pesquisa genômica da década passada, biologia vegetal tem visto apresentando numerosos estudos em larga escala as análises quantitativas de expressão gênica. Microarray e abordagens sequenciamento de última geração estão sendo usados ​​para investigar processos de resposta de desenvolvimento, fisiológicos e stress, dissecar as vias epigenéticas e pequenos RNA, e construir redes de grande gene regulador 1-3. Embora essas técnicas facilitam a análise simultânea de conjuntos de genes de grande porte, que normalmente fornecem uma resolução muito limitado espaço-temporal das mudanças de expressão gênica. Esta limitação pode ser superada parcialmente usando perfis método em conjunto com lasermicrodissection ou fluorescência-ativado celular classificação 4-7. No entanto, para compreender plenamente o papel biológico de um gene, o conhecimento de seu padrão espaço-temporal de expressão em uma resolução celular é essencial. Particularmente, quando se estuda o desenvolvimento ou os efeitos de sti ambientaismuli e mutantes podem a análise detalhada do padrão de expressão de um gene tornam-se essenciais. Por exemplo, sutis diferenças quantitativas nos níveis de expressão de genes-chave de regulamentação pode levar a fenótipos dramática quando associada com a perda ou ganho de expressão, em tipos específicos de células. Vários métodos são usados ​​rotineiramente para a análise detalhada dos padrões de expressão gênica. Uma delas é através da análise das linhas de repórter transgênicos. Tal análise pode, no entanto, tornar-se demorado quando se analisa genes múltiplos ou trabalhando em plantas recalcitrantes a transformação. Além disso, uma validação independente para garantir que o padrão de expressão do transgene imita a do gene endógeno é normalmente necessária. Localização de proteínas imuno-histoquímica ou hibridização in situ mRNA apresentar alternativas relativamente rápido para a visualização direta da expressão do gene nas células e tecidos. Este último tem a vantagem que pode ser facilmente used em qualquer gene de interesse. A hibridização in situ permite a detecção de mRNAs em células-alvo por hibridização com um marcado sentido anti-RNA sonda obtida por transcrição in vitro do gene de interesse. Aqui nós descrevemos um protocolo para a localização em situ da expressão gênica em plantas, que é altamente sensibilidade e específicos. Ele é otimizado para uso com paraformaldeído fixos, parafina seções, que dão excelente preservação da histologia, e sondas DIG-rotulados que são visualizados através da imuno-detecção e fosfatase alcalina-reação colorimétrica. Este protocolo tem sido aplicado com sucesso em uma série de tecidos a partir de uma ampla gama de espécies de plantas, e pode ser usado para analisar a expressão de mRNAs, assim como pequenos RNAs 8-14.

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

O<em&gt; In situ</em&gt; Protocolo de hibridização descrita aqui permite a localização direta de mRNA e expressão do RNA pequeno no nível celular com alta sensibilidade e especificidade. O procedimento é otimizado para parafina cortes de tecidos de plantas, é aplicável a uma grande variedade de plantas e tecidos, e pode ser concluído no prazo de 10 dias.

Hibridização In Situ para a localização precisa de transcrições em Plantas

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

in-situ-hybridization-for-precise-localization-transcripts

Research

JoVE Journal

Biology

In Situ Hybridization for the Precise Localization of Transcripts in Plants

51.1K visualizações.  Cold Spring Harbor Laboratory. O In situ Protocolo de hibridização descrita aqui permite a localização direta de mRNA e expressão do RNA pequeno no nível celular com alta sensibilidade e especificidade. O procedimento é otimizado para parafina cortes de tecidos de plantas, é aplicável a uma grande variedade de plantas e tecidos, e pode ser concluído no prazo de 10 dias.

Vídeo: 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.

Monte Whole dupla hibridização in situ de embriões de galinha precoce

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

Biologia

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.

Fluorescência In situ Hybridization (FISH) Protocolo no esperma humano

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.

Radioativo In situ Hibridação para detecção de padrões de expressão dos genes nos tecidos diversos

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.

Detecção de RNA viral por fluorescência<em&gt; In situ</em&gt; Hybridization (FISH)

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.

Tempo cromossomo Replicando Combinado com Fluorescente<em&gt; Em situ</em&gt; Hibridização

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.

Todo o Monte RNA Fluorescente<em&gt; In situ</em&gt; A hibridação de<em&gt; Drosophila</em&gt; Embriões

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.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</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.

MicroRNA<em&gt; Em situ</em&gt; Hibridização de tecidos de rim fixadas em formalina

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.

Método simples para DNA de Fluorescência<em&gt; In Situ</em&gt; A hibridação com Cromossomos Squashed

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.