Agradecemos Sergei Demin e Tatyana Karamysheva por sua ajuda com a preparação cromossômica e FISH em Anopheles. Agradecemos também a David Severson por nos proporcionar Aedes e Culex DNA genômico BAC clones e Melissa Wade para a edição do texto. Este trabalho foi financiado por duas subvenções dos Institutos Nacionais de Saúde: 1R21 AI88035-01 para Maria V. Sharakhova e 1R21 AI094289-01 para Igor V. Sharakhov.

<ol>
	<li>Sharakhov, I. V., Sharakhova, M. V., Verrity, J. F., Abbington, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromosome Mapping Research Developments. , (2008).</li><li>Holt, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+the+malaria+mosquito+Anopheles+gambiae.">The genome sequence of the malaria mosquito Anopheles gambiae.</a> Science. 298, 129-149 (2002).</li><li>Sharakhova, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+of+the+Anopheles+gambiae+PEST+genome+assembly.">Update of the Anopheles gambiae PEST genome assembly.</a> Genome biology. 8, R5 (2007).</li><li>Campos, J., Andrade, C. F., Recco-Pimentel, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+preparing+polytene+chromosomes+from+Aedes+aegypti+(Diptera,+Culicinae).">A technique for preparing polytene chromosomes from Aedes aegypti (Diptera, Culicinae).</a> Memorias do Instituto Oswaldo Cruz. 98, 387-390 (2003).</li><li>Campos, J., Andrade, C. F., Recco-Pimentel, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malpighian+tubule+polytene+chromosomes+of+Culex+quinquefasciatus+(Diptera,+Culicinae).">Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera, Culicinae).</a> Memorias do Instituto Oswaldo Cruz. 98, 383-386 (2003).</li><li>McAbee, R. D., Christiansen, J. A., Cornel, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+larval+salivary+gland+polytene+chromosome+photomap+for+Culex+quinquefasciatus+(Diptera:+Culicidae)+from+Johannesburg,+South+Africa.">A detailed larval salivary gland polytene chromosome photomap for Culex quinquefasciatus (Diptera: Culicidae) from Johannesburg, South Africa.</a> J. Med. Entomol. 44, 229-237 (2007).</li><li>Nene, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+Aedes+aegypti,+a+major+arbovirus+vector.">Genome sequence of Aedes aegypti, a major arbovirus vector.</a> Science. 316, 1718-1723 (2007).</li><li>Arensburger, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequencing+of+Culex+quinquefasciatus+establishes+a+platform+for+mosquito+comparative+genomics.">Sequencing of Culex quinquefasciatus establishes a platform for mosquito comparative genomics.</a> Science. 330, 86-88 (2010).</li><li>Sharakhova, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaginal+discs--a+new+source+of+chromosomes+for+genome+mapping+of+the+yellow+fever+mosquito+Aedes+aegypti.">Imaginal discs--a new source of chromosomes for genome mapping of the yellow fever mosquito Aedes aegypti.</a> PLoS neglected tropical diseases. 5, e1335 (2011).</li><li>Brown, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+physical+map+of+Aedes+aegypti.">Toward a physical map of Aedes aegypti.</a> Insect Mol. Biol. 4, 161-167 (1995).</li><li>Steiniger, G. E., Mukherjee, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+chromosome+banding:+technique+for+G-+and+Q-banding+pattern+in+the+mosquito+Aedes+albopictus.">Insect chromosome banding: technique for G- and Q-banding pattern in the mosquito Aedes albopictus.</a> Can. J. Genet. Cytol. 17, 241-244 (1975).</li><li>Garimberti, E., Tosi, S., Bridger, J. M., Volpi, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence in situ hybridization (FISH). , (2010).</li><li>Scott, J. A., Brogdon, W. G., Collins, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+single+specimens+of+the+Anopheles+gambiae+complex+by+the+polymerase+chain+reaction.">Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction.</a> The American journal of tropical medicine and hygiene. 49, 520-529 (1993).</li><li>Trifonov, V. A., Vorobyeva, N. N., Rens, W., Leiehr, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence in situ hybridization (FISH). , (2009).</li><li>Collins, F. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ribosomal+RNA+gene+probe+differentiates+member+species+of+the+Anopheles+gambiae+complex.">A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex.</a> The American journal of tropical medicine and hygiene. 37, 37-41 (1987).</li><li>Kumar, A., Rai, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+localization+and+copy+number+of+18S+28S+ribosomal+RNA+genes+in+evolutionary+diverse+mosquitoes+(Diptera,+Culicidae).">Chromosomal localization and copy number of 18S+28S ribosomal RNA genes in evolutionary diverse mosquitoes (Diptera, Culicidae).</a> Hereditas. 113, 277-289 (1990).</li><li>Brown, S. E., Knudson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+landmarks+for+Aedes+aegypti+chromosomes.">FISH landmarks for Aedes aegypti chromosomes.</a> Insect Mol. Biol. 6, 197-202 (1997).</li><li>Brown, S. E., Severson, D. W., Smith, L. A., Knudson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+the+Aedes+aegypti+mosquito+genetic+linkage+and+physical+maps.">Integration of the Aedes aegypti mosquito genetic linkage and physical maps.</a> Genetics. 157, 1299-1305 (2001).</li></ol>

Não há conflitos de interesse declarados.

Não fluorescente na hibridação in situ em cromossomos mitóticos dos mosquitos foi realizada pela primeira vez em 1990 por A. Kumar e Rai K. 17. Nesse estudo, 18S e 28S do DNA ribossomal, clonados juntos em um plasmídeo, foram colocadas para os cromossomas de 20 espécies de mosquitos. A sonda de ADN foi marcada radioactivamente e hibridada com os cromossomas de gânglios cérebro. Entre três gêneros de mosquitos, uma técnica de FISH foi desenvolvido apenas para cromossomos mitóticos a partir da linha de células de Ae. aegypti 10,18,19 e nunca foi realizada em cromossomos mitóticos de mosquitos vivos. Recentemente, foi desenvolvida uma técnica simples e robusta para a obtenção de preparações de alta qualidade dos cromossomos de IDs de 4 larvas ínstares 9. Este método permite que um grande número de cromossomas para ser obtido de uma corrediça e pode ser universalmente utilizado para todas as espécies de mosquitos. A necessidade de utilizar apenas sta não larval, pupal ou adultoges de mosquitos, para preparação da lâmina é provavelmente a única limitação do método. O método padrão FISH 12 foi otimizado para uso genômica BAC clone e IGS rDNA como sondas para os cromossomos mitóticos de Aedes, Culex e Anopheles. Para além destas aplicações específicas, os protocolos de FISH descritas aqui podem também ser usados ​​para outros fins. O protocolo de FISH avançado, que utiliza fracções de ADN C 0 t para bloquear a hibridização não específica, também pode ser aplicado para a hibridação de clones BAC ou quaisquer outros fragmentos de DNA em grandes regiões de Anopheles heterocromatina. Regiões heterocromáticas são enriquecidas com elementos transponíveis e repete outros, e sondas dessas regiões produzem normalmente sólida sobre os três cromossomos. Usando Unlabeled C 0 frações de DNA t vai ajudar a reduzir a hibridação inespecífica da sonda para os cromossomos. A versão simples do FISH protocol pode ser usado para qualquer rDNA ou as sondas de ADN repetitivo em cromossomos mitóticos de mosquitos e outros insectos. Além disso, também pode ser aplicado para a hibridação de DNA clone BAC em espécies com baixo teor de ADN repetitivo em regiões totalmente eucromático como Anopheles ou Drosophila. O protocolo proposto aqui vai ajudar a obter altamente acabados assembléias cromossomo base do genoma do mosquito e pode ser amplamente utilizado para aplicações de citogenética vários outros grupos de insetos.

<table border="1"><tbody><tr><td> Nome do reagente / Equipamentos </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> MZ6 estereomicroscópio Leica </td><td> Leica </td><td> VA-OM-E194-354 </td><td> Um estereomicroscópio diferente pode ser usado </td></tr><tr><td> Olympus CX41 microscópio de fase </td><td> Olimpo </td><td> CX41 </td><td> Um microscópio de fase diferente pode ser usado </td></tr><tr><td> Olympus BX61 microscópio fluorescente </td><td> Olimpo </td><td> BX61 </td><td> Um microscópio fluorescente diferente pode ser usado </td></tr><tr><td> Deslize ThermoBrite Sistema de Desnaturação / Hibridização </td><td> Abbott Molecular </td><td> 30-144110 </td><td> Serve como um bloco de aquecimento e uma câmara úmida </td></tr><tr><td> Agulhas de dissecação </td><td> ScienceTools Belas </td><td> 10130-10</td><td></td></tr><tr><td> Porta agulhas </td><td> Belas Science Tools </td><td> 26018-17 </td><td></td></tr><tr><td> Tesouras de dissecação </td><td> Belas Science Tools </td><td> 15000-03 </td><td></td></tr><tr><td> 75x25 dupla fosco micro desliza </td><td> Corning </td><td> 2949-75x25 </td><td></td></tr><tr><td> 22x22 mm lamínulas de microscópio </td><td> Fisher Scientific </td><td> 12-544-10 </td><td></td></tr><tr><td> Parafilm </td><td> Fisher Scientific </td><td> 13-374-10 </td><td></td></tr><tr><td> Borracha Cimento </td><td> Fisher Scientific </td><td> 50-949-105 </td><td></td></tr><tr><td> Ácido acético </td><td> Fisher Scientific </td><td> A491-212 </td><td></td></tr><tr><td> Prova de álcool 200 </td><td> Decon Laboratories </td><td> 2701 </td><td></td></tr><tr><td> Ácido propiônico </td><td> Sigma-Aldrich </td><td> 402907 </td><td></td></tr><tr><td> Ácido clorídrico </td><td> Fisher Scientific </td><td> A144-500 </td><td></td></tr><tr><td> Di-hidrato de citrato de sódio </td><td> Fisher Scientific </td><td> S279-500 </td><td></td></tr><tr><td> Tri-hidrato de acetato de sódio </td><td> Fisher Scientific </td><td> BP334-500 </td><td></td></tr><tr><td> Cloreto de potássio </td><td> Fisher Scientific </td><td> BP366-500 </td><td></td></tr><tr><td> EDTA </td><td> Fisher Scientific </td><td> S311-500 </td><td></td></tr><tr><td> Tris base </td><td> Fisher Scientific </td><td> BP152-1 </td><td></td></tr><tr><td> PBS 10x </td><td> Invitrogen </td><td> P5493 </td><td></td></tr><tr><td> 10% NBF (formalina tamponada neutra) </td><td> Sigma-Aldrich </td><td> HT501128 </td><td></td></tr><tr><td> Formamida 99% </td><td> Fisher Scientific </td><td> BP227500 </td><td></td></tr><tr><td> Sal de sódio de sulfato de dextrano </td><td> Sigma-Aldrich </td><td> D8906 </td><td></td></tr><tr><td> 20x tampão SSC </td><td> Invitrogen </td><td> AM9765 </td><td></td></tr><tr><td> 1 mM de iodeto de YOYO-1 (491/509) de solução </td><td> Invitrogen </td><td> Y3601 </td><td></td></tr><tr><td> Antifade Prolongar reagente Ouro </td><td> Invitrogen </td><td> <a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=P36930" target="_blank">P36930</a> </td><td></td></tr><tr><td> dATP, dCTP, dGTP, dTTP </td><td> Fermentas </td><td> R0141, R0151, R0161, R0171 </td><td></td></tr><tr><td> Cy3-dUTP, Cy5-dUTP </td><td> GE Healthcare </td><td> PA53022, PA55022 </td><td></td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> A3294 </td><td></td></tr><tr><td> DNA Polimerase I </td><td> Fermentas </td><td> EP0041 </td><td></td></tr><tr><td> DNase I </td><td> Fermentas </td><td> EN0521 </td><td></td></tr><tr><td> S1 nuclease </td><td> Fermentas </td><td> EN0321 </td><td></td></tr><tr><td> Taq DNA Polymerase </td><td> Invitrogen </td><td> 18038-042 </td><td></td></tr><tr><td> RNase </td><td> Sigma-Aldrich </td><td> 9001-99-4 </td><td></td></tr><tr><td> Pepsina </td><td> USB </td><td> 9001-75-6 </td><td></td></tr><tr><td> ADN de esperma de salmão </td><td> Sigma-Aldrich </td><td> D7656 </td><td></td></tr><tr><td> Nonidet-P40 (NP40) </td><td> EUA Biológica </td><td> NC9375914 </td><td></td></tr><tr><td> Qiagen Blood and Maxikit Cultura Celular </td><td> Qiagen </td><td> 13362 </td><td></td></tr><tr><td> Qiagen Kit construir grandes </td><td> Qiagen </td><td> 12462 </td&gt; <td></td></tr></tbody></table>

fluorescent in situ hybridization on mitotic chromosomes of mosquitoes

Hibridação fluorescente in situ (FISH) é uma técnica habitualmente usada por muitos laboratórios para determinar a posição cromossómica de sondas de DNA e RNA. Uma importante aplicação do presente método é o desenvolvimento de mapas de alta qualidade físicas úteis para melhorar os conjuntos do genoma de vários organismos. O padrão natural de bandas dos cromossomos politênicos e mitóticos fornece orientação para o ordenamento preciso e orientação dos supercontigs genômicos. Dentre os três gêneros de mosquitos Anopheles, ou seja, Aedes, Culex e, uma bem estabelecida técnica de mapeamento baseado cromossomo foi desenvolvido apenas para Anopheles, cujos membros possuem cromossomos politênicos legíveis 1. Como resultado dos esforços de mapeamento do genoma, 88% da Uma. gambiae genoma foi colocado para posições precisas cromossoma 2,3. Dois gêneros de mosquitos Aedes outros, e Culex, que mal polytenized b cromossomosomo de super-representação significativa de elementos transponíveis em seus genomas 4, 5, 6. Apenas 31 e 9% dos supercontings genômicas foram atribuídos sem ordem ou orientação de cromossomos de Ae. aegypti 7 e CX. quinquefasciatus 8, respectivamente. Preparação mitótico cromossoma para estas duas espécies tinham sido previamente limitado aos gânglios cerebral e linhas celulares. No entanto, as lâminas cromossoma preparadas a partir de gânglios cérebro de mosquitos geralmente contêm baixos números de placas de metafase 9. Além disso, embora a técnica FISH foi desenvolvido para cromossomas mitóticos a partir de uma linha de células de Ae. aegypti 10, a acumulação de múltiplos rearranjos cromossômicos em cromossomos das células da linha 11 torna inútil para mapeamento do genoma. Aqui descrevemos uma técnica simples e robusta para a obtenção de alta qualidade de preparações cromossômicas mitóticas de discos imaginais (IDs) de 4 larvas ínstares, que cum ser utilizado para todos os três géneros de mosquitos. Um protocolo de FISH padrão de 12 é otimizado para uso de clones BAC de DNA genômico como uma sonda em cromossomos mitóticos de Ae. aegypti e Cx. quinquefasciatus., e para a utilização de um espaçador intergênica (IGS) região do DNA ribossomal (rDNA) como uma sonda em um. cromossomas gambiae. Além de mapeamento físico, a técnica desenvolvida pode ser aplicada a citogenética de populações e taxonomia cromossoma / sistemática de mosquitos e grupos de outros insectos.

Watch this Scientific Journal Video about Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes at JoVE.com

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Dentre os três gêneros de mosquito, ou seja,<em&gt; Anopheles,</em&gt;<em&gt; Aedes</em&gt;, E<em&gt; Culex</em&gt;, Técnicas de mapeamento do genoma físicas foram estabelecidos apenas para<em&gt; Anopheles</em&gt;, Cujos membros possuem cromossomos politênicos legíveis. Para os gêneros de<em&gt; Aedes</em&gt; E<em&gt; Culex</em&gt; No entanto, o mapeamento de citogenética permanece desafiador por causa da má qualidade dos cromossomos politênicos. Aqui apresentamos um protocolo universal para a obtenção de alta qualidade preparações de cromossomos mitóticos e um protocolo FISH otimizado para os três gêneros de mosquitos.

Fluorescente<em&gt; In situ</em&gt; Hibridação em cromossomos mitóticos de mosquitos

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

fluorescent-in-situ-hybridization-on-mitotic-chromosomes-of-mosquitoes

Research

JoVE Journal

Immunology and Infection

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

25.6K visualizações.  Virginia Tech. Dentre os três gêneros de mosquito, ou seja, Anopheles, Aedes, E Culex, Técnicas de mapeamento do genoma físicas foram estabelecidos apenas para Anopheles, Cujos membros possuem cromossomos politênicos legíveis. Para os gêneros de Aedes E Culex No entanto, o mapeamento de citogenética permanece desafiador por causa da má qualidade dos cromossomos politênicos. Aqui apresenta...

Vídeo: Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinação de adesivos de fita baseada em amostragem e de fluorescência In situ Hibridação para detecção rápida de Salmonella On Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Imunologia e Infecção

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Fluxo de ácido nucléico bloqueado Citometria de fluorescência In situ Hibridação (LNA fluxo FISH): um método para detecção de RNA bacteriano Pequenas

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Hibridização<em&gt; In situ</em&gt; Das glândulas salivares, ovários e embriões de mosquitos vetores

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Usando proteínas fluorescentes para visualizar e Quantificar<em&gt; Chlamydia</em&gt; Vacúolo Crescimento Dynamics em células vivas

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Pintura de cromossomo 2D e 3D em mosquitos da malária

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Amostragem não-invasiva de mucosa líquido para a quantificação dos níveis de imune-mediador na Vivo das vias aéreas superiores

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

Hibridação in situ da fluorescência quantitativa (peixes) e immunofluorescence (se) de produtos específicos do gene em pilhas KSHV-infectadas

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Visualização de candida albicans no trato gastrointestinal murina usando fluorescente in situ hibridização

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

Análises de competência vetorial sobre mosquitos Aedes aegypti que usam zika vírus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Propagação do Parasita Microsporidiano Edhazardia aedis em Mosquitos Aedes aegypti

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...