Agradecemos Sacha Reichman e Dominique Santiard-Baron pela ajuda na edição do protocolo de purificação RNA.

1. Jornal: Procedimento para recuperar as amostras do Bloco Cirúrgico No laboratório <ol><li> Obter um contrato de importação da empresa de transporte expresso. </li><li> Preencha 10 (ou 25) formas de envio com o endereço postal do laboratório, indicando a pessoa de contato em laboratório (número de telefone e endereço de e-mail). </li><li> Prepare a 10 (ou 25) em forma de amostras numeradas de 1 a 10 (ou 25). Estas formas incluem espaços dedicados a informar sobre a) identificação anônima do paciente, b) a data da cirurgia e c) quaisquer observações adicionais, o cirurgião gostaria de adicionar. Prepare a 10 (ou 25) envelopes almofadados (150 x 210 mm). </li><li> Preparado utilizando ARNase isento de reagentes 25 (ou 65) ml de cloreto de guanidina 6 M em dietilpirocarbonato (DEPC) tratado com H 2 O (GHCL). </li><li> Encher 10 ou 25 numerados de 5 ml de fundo redondo de polietileno tubos estéreis com 2,4 ml de solução GHCL. </li><li> Usado gás árgon para preencher a parte de cima destes tubos, que prima tightly na tampa para evitar a oxidação da solução GHCL ao longo de vários anos de armazenagem no gabinete cirúrgico. </li><li> Introduzir a cada 5 ml tubos em um ml de polipropileno tubo de fundo cônico 50 estéril com tampa de rosca. Use um pedaço de lenço de papel limpo para segurar o tubo de 5 ml para o tubo de 50 ml, fechar o tubo. </li><li> Coloque os tubos de 50 ml em um rack de poliestireno e do rack em uma caixa de papelão com os envelopes almofadados, as formas de transporte, as formas de amostras. </li><li> Cole as instruções (veja: 1,12-1,19) no interior da tampa da caixa de papelão para facilitar a sua leitura. </li><li> Enviar a caixa de papelão pelo correio para a pessoa de contato no hospital. </li></ol> No hospital <ol start="11"><li> Traga a caixa de papelão do gabinete cirúrgico para a sala cirúrgica. Cuidado Nota: Se o procedimento envolve um paciente imunológico comprometido, o papelão não devem ser trazidos para a sala cirúrgica. </li><li> Durante a cirurgia, coloque o SPECIM retinaen numerado em 5 ml de polipropileno estéril preenchido com solução GHCL. Feche bem o tubo. </li><li> Retorne o tubo rapidamente. </li><li> Coloque o tubo sobre o roqueiro tubo de sangue por 10 min. </li><li> Preencha o formulário numerado amostra que o acompanha. </li><li> Informar o número de identificação para a numeração 50 ml tubo correspondente. </li><li> Introduzir o tubo de 5 ml com o espécime cirúrgico no tubo de 50 ml, substituir o tecido de papel e aperte o tubo. </li><li> Introduzir o tubo e a forma de exemplo para encher em um dos envelopes almofadados anexas. Fechá-lo. </li><li> Ligue para a empresa de transporte expresso para pegar. </li></ol> 2. Purificação de RNA No laboratório <ol><li> Homogeneizar a amostra em seu 5 ml de polipropileno tubo com a solução GHCL com um homogeneizador de 1 min enquanto se movimenta o tubo de cima e para baixo. </li><li> Adicionar 270 ul de 2 M de acetato de potássio pH 5,0. Agita-se vigorosamente durante 10 min, colocando o tubo num agitador na suaposição horizontal (420 min -1). </li><li> Centrifugar 10 min a 5000 rpm (6500 xg) a 20 ° C. </li><li> Aspirar suavemente a fração solúvel, sem perturbar o sedimento. </li><li> Transfira para um tubo estéril de 14 ml. </li><li> Adicionar 5,3 mL de Tris 100 mM pH 8,0, N-lauroilsarcosina a 1%. </li><li> Adicionar 3,2 g de cloreto de césio (CsCl) e o tubo de misturar em vortex. </li><li> Adicionar 1,8 ml de ácido de CsCl / ácido etilenodiaminotetracético (EDTA) num tubo de centrífuga de polialómero ml estéril 11. </li><li> Com 10 ml estéril pipeta Pasteur, transferir a solução de ARN para 1,8 ml de CsCl / EDTA, deslizando-se lentamente sobre a borda do tubo, para evitar perturbar a almofada de densidade. </li><li> Colocar os tubos (um segundo tubo contendo os buffers sem retina, se necessário) no rotor. </li><li> Centrifugar 24 horas a 32.000 rpm (225.000 xg) a 20 ° C. </li><li> Remova a parte superior da solução com uma pipeta Pasteur estéril, e descartá-lo. </li><li> Retire gradualmente durante a verificação damomento em que o ADN (viscoso) é aspirado com uma pipeta de Pasteur estéril, segundo, e descartá-lo. </li><li> Remover a solução restante tendo o cuidado de não libertar o sedimento de ARN com uma terceira pipeta Pasteur estéril. </li><li> A seção inferior do tubo com um bisturi chama-esterilizados, em seguida, colocar o restante do tubo de cabeça para baixo em um olhar estéril. </li><li> Inverta o tubo e lave delicadamente com 160 ml de GHCL. </li><li> Deixar o sedimento secar durante 10 min. </li><li> Ressuspender o sedimento em 150 ul de (Tris 10 mM pH 7,5 - EDTA 1 mM - 0,1% de SDS). </li><li> Transferir a solução para um tubo de microcentrífuga estéril ml 2, depois colher o sedimento residual com 30 ul de (Tris 10 mM pH 7,5 - EDTA 1 mM - 0,1% de SDS). </li><li> Adicionar 150 ul de (Tris 10 mM pH 7,5 - EDTA 1 mM). </li><li> Adicionar 30 ul de 3 M de acetato de sódio pH 5.0, o tubo de vórtice. </li><li> Adicionar 900 ul de etanol a 100% (-20 ° C), o tubo de vórtice. </li><li> Colocar o tubo de 30 min em gelo fundente. </li><li> Centrifuge o tubo 30 min a 15.000 rpm a 4 ° C. </li><li> Aspirar delicadamente a fração solúvel, e descartá-lo. </li><li> Adicionar 500 ul de etanol a 70% (RT), o tubo de vórtice. </li><li> Centrifugar o tubo de 20 min a 15.000 rpm (18.000 xg) a 4 ° C. </li><li> Repetir o passo de enxaguamento (70% de etanol). </li><li> Centrifugar brevemente e eliminar o etanol remanescente com uma pipeta P200. </li><li> Deixar o sedimento secar durante 10 min. </li><li> Ressuspender o sedimento em 50 ul tratada com DEPC H2O </li><li> Misture vigorosamente em vortex. </li><li> Incubar 15 min a 45 ° C num banho de água. </li></ol> 3. Análise de RNA por eletroforese em gel <ol><li> Despeje um gel de agarose dentro de um capuz química. </li><li> Num tubo de microcentrífuga de 1,5 ml estéril, adicionar 2 ul de RNA a ser analisado e de 6,4 mL de tampão de preparação de amostra. </li><li> Num segundo tubo de 1,5 ml, adicionar 3 ml de padrões de RNA e de 9,6 mL de tampão de preparação de amostra. </li><li> Aquece-se a tubos de 15 min a 65 ° C, Colocá-los no gelo. </li><li> Adicionar 1 mL de brometo de etidio (EB), tampão de carregamento sem corante no tubo de amostra de ARN e 1 ul de tampão EB carga com corante no tubo de padrões de ARN. </li><li> Executar o gel inferior a 80 - 100 V dentro de uma capa de química em tampão de funcionamento, até que um dos corantes (azul de bromofenol) atinge 2/3 do fundo do gel. </li><li> Lavar o gel duas vezes, 15 min, com 250 ml de tratada com DEPC 2x SSC. </li><li> Pegue uma imagem digitalizada sob iluminação UV. </li><li> Calcular a relação entre a banda superior (23S rRNA) e a banda inferior (ARNr 16S). </li></ol> 4. A purificação final do ARN <ol><li> Ajustar o volume da amostra de RNA com 100 ul com tratada com DEPC H2O (se necessário). </li><li> Adicionam-se 100 ul de ácido fenol (1 ml de fenol saturado em tratada com DEPC H2O + 130 ul de 50 mM de acetato de sódio, pH 5,2), vortex vigorosamente. </li><li> Centrifugar a 10 min a 13.000 rpm (15.000 x g), à temperatura ambiente. </li><li> Recover e transferir cuidadosamente a fase aquosa (fase superior) para um novo um tubo de microcentrífuga de 1,5 ml estéril. </li><li> Adicionar 10 ul de acetato de sódio 3M, pH 5,2 e 250 ul de etanol a 100% (-20 ° C). </li><li> Incubar o tubo 30 min em gelo fundente. </li><li> Centrifugar o tubo de 30 min a 14.000 rpm (18.000 xg) a 4 ° C. </li><li> Aspirar cuidadosamente a fração solúvel, e descartá-lo. </li><li> Adicionar 500 ul de etanol a 70% (temperatura ambiente), e o tubo de vórtice. </li><li> Centrifugar o tubo de 20 min a 14.000 rpm (18.000 xg) a 4 ° C. </li><li> Repetir o passo de enxaguamento (70% de etanol). </li><li> Centrifugar brevemente e eliminar o etanol remanescente com uma pipeta P200. </li><li> Deixe o ar seco do sedimento para 10 min. </li><li> Ressuspender o sedimento em 50 mL de tratada com DEPC H2O </li><li> Incubar em seguida, tubo de 15 min a 45 ° C. </li><li> Medir a absorvância (Abs) a 260 nm e 280 nm, em 2 mL da amostra de RNA. Calculado o Abs260/Abs280 ratio. </li> &lt;li&gt; loja a amostra de ARN a -80 ° C (estável durante vários anos). </li></ol> 5. Soluções e procedimentos de limpeza <ol><li> Acetato de potássio 2 M, pH 5,0: Dissolver 19,6 g de acetato de potássio em 70 ml de DEPC-H2O tratada Limpar a sonda medidor de pH por imersão em com NaOH 1M durante 15 min, seguido de 5 lavagens com tratada com DEPC H2O Ajustar a pH 5,0 com ácido acético, em seguida, ajustar o volume para 100 ml com DEPC-H2O tratada </li><li> Tris-HCl 100 mM, pH 8,0, N-lauroilsarcosina 1%: Dissolve-se 2 g de N-lauroilsarcosina (sob um capuz) para 40 ml de 0,5 M de Tris base, pH 8,0. Ajustar o volume a 200 ml com DEPC-H2O tratada) de CsCl / EDTA: 96 g de CsCl em 50 ml de 10 mM EDTA pH 7,5, preparado com DEPC-H2O tratada Autoclave e ajustar o volume a 100 ml com tratada com DEPC H2O Verificar pH &lt;7,5. </li><li> Em gel de agarose 1%: Coloque 1 g de agarose em 62 ml de DEPC-H2O tratada Coza no forno de microondas. Adicionar 18 ml de 12,3 Mformaldeído, 20 ml de MOPS 5x. Verter dentro de uma capa de química. </li><li> Preparação de amostras de buffer: Para ser extemporânea preparado. Misturar no interior de um produto químico cobertura 8 ul de MOPS 5x, 16 ml de formaldeído a 12,3 M e 40 ul de formamida. </li><li> Tampão de carregamento EB (sem corante): Para ser extemporânea preparado. Adicionar 4 ul de 10 mg / ml de brometo de etídio em 20 ul de glicerol a 80% preparada com tratada com DEPC H2O </li><li> Tampão de carregamento EB (com corante): Para ser extemporânea preparado. Adicionam-se 2 ul de 10 mg / ml de brometo de etídio em 10 ul de glicerol a 80% contendo 0,25% de xilema cianol, 0,25% de azul de bromofenol tratada com DEPC H2O </li><li> O tampão de corrida: A 60 ml de MOPS 5x, adicionar 54 mL de 12,3 M de formaldeído e 186 ml de tratada com DEPC H2O </li><li> Tratada com DEPC H2O: água destilada Incubar com 0,1% v / v de dietilpirocarbonato durante 2 horas a 37 ° C com agitação. Esterilizar em autoclave. </li><li> Limpando o homogeneizador: Adicionar 25 ml de solução de limpeza (RBS) para 1 L de DEPCH2O tratada, e imergir o eixo 1 min com agitação. Incubar 2 horas a 60 ° C. Enxaguar abundantemente com DEPC tratados com H 2 O Enrole o instrumento em alumínio e colocar na caixa do instrumento. Embrulhe a caixa. Esterilizar em autoclave. </li><li> A limpeza do dispositivo de electroforese: Lavar o aparelho, a bandeja e o pente de 15 poços. Incubar 15 minutos em 3% de peróxido de hidrogénio. Lavar uma vez com etanol a 100%. Ar seco. </li><li> A limpeza dos pratos de vidro: Limpe os pratos de vidro RBS 2% durante toda a noite, em seguida, enxaguar com água destilada antes da esterilização a 200 ° C. </li></ol>

<ol>
	<li>Mitry, D., Charteris, D. G., 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+epidemiology+of+rhegmatogenous+retinal+detachment:+geographical+variation+and+clinical+associations.">The epidemiology of rhegmatogenous retinal detachment: geographical variation and clinical associations.</a> The British Journal of Ophthalmology. 94 (6), 678 (2010).</li><li>Green, R. E., Krause, J., 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+draft+sequence+of+the+Neandertal+genome.">A draft sequence of the Neandertal genome.</a> Science. 328 (5979), 710 (2010).</li><li>Glisin, V., Crkvenjakov, R., 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=Ribonucleic+acid+isolated+by+cesium+chloride+centrifugation.">Ribonucleic acid isolated by cesium chloride centrifugation.</a> Biochemistry. 13 (12), 2633 (1974).</li><li>Van Gelder, R. N., von Zastrow, M. 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=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 87 (5), 1663 (1990).</li><li>Cavalieri, L. F., Yamaura, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=coli+tRNAs+as+inhibitors+of+viral+reverse+transcription+in+vitro.">coli tRNAs as inhibitors of viral reverse transcription in vitro.</a> Nucleic Acids Research. 2 (12), 2315 (1975).</li><li>Sawadogo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+inhibition+of+yeast+RNA+polymerases+A+and+B+by+tRNA+and+alpha-amanitin.">On the inhibition of yeast RNA polymerases A and B by tRNA and alpha-amanitin.</a> Biochemical and biophysical research communications. 98 (1), 261 (1981).</li><li>Delyfer, M. N., Raffelsberger, W., 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=Transcriptomic+analysis+of+human+retinal+detachment+reveals+both+inflammatory+response+and+photoreceptor+death.">Transcriptomic analysis of human retinal detachment reveals both inflammatory response and photoreceptor death.</a> PloS ONE. 6 (12), e28791 (2011).</li><li>Kalathur, R. K., Gagniere, N., 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=RETINOBASE:+a+web+database,+data+mining+and+analysis+platform+for+gene+expression+data+on+retina.">RETINOBASE: a web database, data mining and analysis platform for gene expression data on retina.</a> BMC Genomics. 9, 208 (2008).</li><li>Nakazawa, T., Hisatomi, T., 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=Monocyte+chemoattractant+protein+1+mediates+retinal+detachment-induced+photoreceptor+apoptosis.">Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (7), 2425 (2007).</li><li>Leveillard, T., Sahel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod-derived+cone+viability+factor+for+treating+blinding+diseases:+from+clinic+to+redox+signaling.">Rod-derived cone viability factor for treating blinding diseases: from clinic to redox signaling.</a> Science Translational Medicine. 2 (26), 26ps16 (2010).</li><li>Chalmel, F., Leveillard, T., 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=Rod-derived+Cone+Viability+Factor-2+is+a+novel+bifunctional-thioredoxin-like+protein+with+therapeutic+potential.">Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential.</a> BMC Molecular Biology. 8, 74 (2007).</li></ol>

Os autores não têm nada a revelar.

O desenvolvimento de um processo para a recuperação do tecido a partir do bloco cirúrgico tem sido essencial para a análise do transcriptoma de descolamento da retina. Deve-se notar que este tipo de cirurgia é praticada em situações de emergência e que o oftalmologista operacional têm pouco tempo para participar de um programa de pesquisa biológica quando operam. Este é também realizada retinectomy estocasticamente em cada serviço, de modo que a maneira mais fácil de atingir números estatísticos é traba...

O principal objectivo terapêutico no descolamento da retina (DR) é a de encontrar uma maneira para limitar os danos de células fotorreceptoras da retina e inflamação resultante da separação dos fotorreceptores a partir das células epiteliais pigmentadas da retina. Durante RD, células de RPE são activados, migrar desdiferenciem, e proliferar na superfície da retina descolada, exercendo forças contrácteis levando a complicações. Análise transcriptômica de RD é uma maneira de identificar genes-alvo com expressão alterada na sequência RD e, portanto, futuros moléculas terapêuticas que poderiam melhorar o resultado visual final em combinação com a cirurgia. É bem sabido que o ácido ribonucleico (RNA) não é estável como é o ácido desoxirribonucleico (ADN), o último sendo amplamente utilizados para estudos genéticos, a sua estabilidade tinha permitido a sequenciação do genoma a partir de amostras neandertalense paleontológicos de mais de 30.000 anos de idade 2. A transcrição do DNA dos genes a partir do genoma em RNA mensageiro é oimportante no processo de expressão de genes, e o mRNA é instável, a fim de constituir um sinal. RNAs são muito rapidamente degradados pelas enzimas ARNase que terminam o sinal. Quando são isoladas a partir de tecidos de um organismo, os RNAs são muitas vezes degradados antes que a expressão é estudado num laboratório de investigação. RNA degradado não é adequado para análise de expressão génica. Porque o pessoal do laboratório não pode participar de cirurgia, foi desenvolvido um procedimento que é fácil e requer apenas que o cirurgião para recuperar os tecidos em uma solução de RNase apropriado. O ARN dos tecidos é estável e pode ser analisado sem qualquer sinal de desgaste, após 72 horas à temperatura ambiente nesta solução. Os ARN provenientes de amostras são purificados por um método normalizado que envolve uma ultracentrifugação em gradiente de cloreto de césio depois de ter sido transferido para o laboratório 3. Em seguida, a qualidade dos ARN são avaliados por electroforese em gel de agarose e por RT-PCR. O protocolo de purificação de RNA tem ovantagem de separar as moléculas de acordo com a sua densidade, que é diferente do DNA e RNA e assegura que o ARN não é contaminada por uma moléculas de ADN que irão gerar sinais artefatuais em estudos de expressão de genes. Além disso, os ARN de transferência (ARNt), que são quantitativamente o ARN mais abundantes no interior de uma célula, são separados de acordo com as mesmas propriedades físicas do RNA ribossomal (rRNAs) e o RNA mensageiro (mRNA), os dois últimos sendo um deles o o produto final do processo de purificação. A remoção de ARNt a partir da preparação é útil uma vez que a maioria dos protocolos analíticos microarray envolveu a utilização de transcriptase reversa e ARN polimerases que são inibidos pelo ARNt 4-6. O ARN purificado a partir de amostras cirúrgicas são rotulados usando o protocolo padrão e hibridado com um chip de microarray, e os resultados são analisados ​​utilizando dois métodos complementares, o método de taxa de detecção falsa, e utilizando um novo método baseado na informação mútua e visualized no servidor web-based Retinobase 7,8.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Aventi J-E</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotor</td>
 <td>Beckman Coulter</td>
 <td>JS-5.3</td>
 <td></td>
 </tr>
 <tr>
 <td>Ultracentrifuge</td>
 <td>Beckman Coulter</td>
 <td>LE 80K</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotor</td>
 <td>Beckman Coulter</td>
 <td>SW41</td>
 <td></td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>Biorad</td>
 <td>Pac 3000</td>
 <td></td>
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 <td>PolytronTM</td>
 <td>Kinematica</td>
 <td>PT 2100</td>
 <td>Supplied with PT-DA 2105:2</td>
 </tr>
 <tr>
 <td>Agarose gel electrophoresis device</td>
 <td>Biorad</td>
 <td>MiniGel Cell GT</td>
 <td></td>
 </tr>
 <tr>
 <td>Imaging System </td>
 <td>Biorad</td>
 <td>GelDoc-It Imager</td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml sterile polyethylene tube</td>
 <td>Greiner</td>
 <td>115261</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile polyallomer centrifuge tube </td>
 <td>Beckman Coulter</td>
 <td>331372</td>
 <td></td>
 </tr>
 <tr>
 <td>Chemical reagents</td>
 <td>Sigma</td>
 <td></td>
 <td>Molecular biology grade (RNase and DNase free products)</td>
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 <tr>
 <td>Cleaning Solution </td>
 <td>VWR </td>
 <td>RBS</td>
 <td></td>
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 <tr>
 <td>Microarray chip</td>
 <td>Affymetrix</td>
 <td>Human U133 plus 2 array</td>
 <td></td>
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</table>

transcriptomic analysis of human retinal surgical specimens using journal

Descolamento da retina (RD) descreve a separação da retina neurosensorial do epitélio pigmentado da retina (RPE). O RPE é essencial para a função normal dos neurônios sensíveis à luz, os fotorreceptores. Descolamento de retina, a partir do RPE cria uma lacuna física que é preenchido com o fluido extracelular. RD inicia os eventos adversos celulares e moleculares que afectam tanto a retina neurosensorial e o RPE desde a troca fisiológico de iões e metabolitos é gravemente perturbada. A consequência de visão está relacionada com a duração do desprendimento desde um reapposition rápida dos dois tecidos resulta na restauração da visão 1. O tratamento de RD é exclusivamente cirúrgico. A remoção do gel vítreo (vitrectomia) é seguido pela remoção de parte não essencial da retina em torno da área individual para favorecer descolamento da retina. As amostras retiradas da retina são res nullius (nada) e, conseqüentemente, normalmente descartado.Para recuperar RNA desses espécimes cirúrgicos, desenvolvemos o processo diário que permite a conservação da RNA durante a transferência do bloco cirúrgico para o laboratório. Também um protocolo padronizado para purificar RNA por ultracentrifugação de cloreto de césio para assegurar que os ARN purificados são adequados para análise da expressão genética global. A qualidade do RNA foi validada tanto por RT-PCR e análise de microarray. A análise dos dados mostra uma participação simultânea da inflamação e degeneração de fotorreceptores durante RD.

O procedimento de revista (Figura 1) permite-nos recuperar amostras de retina do bloco cirúrgico para RNA purificado, e analisar o transcriptoma de descolamento de retina. Descolamento da retina resultados na regulação positiva do gene quimiotática de monócitos MCP1 CCL2, e na diminuição da expressão do gene da haste de fotorreceptores transdução GNAT1, o cone de ondas curtas opsina OPN1SW eo homeogene CRX (Figura 2). A indução de CCL2...

Watch this Scientific Journal Video about Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl at JoVE.com

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Usamos amostras de retina de retinectomy para uma análise transcriptomic de descolamento de retina. Foi desenvolvido um processo que permite a conservação de ARN entre os blocos cirúrgicos e laboratoriais. Temos um protocolo padronizado para purificar RNA por ultracentrifugação de cloreto de césio para assegurar que os ARN purificados são adequados para a análise de microarray.

Análise de Transcriptoma Humanos espécimes cirúrgicos retina utilizando Jornal

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

transcriptomic-analysis-human-retinal-surgical-specimens-using

Research

JoVE Journal

Medicine

7.7K visualizações.  Institut National de la Santé et de la Recherche Médicale. Usamos amostras de retina de retinectomy para uma análise transcriptomic de descolamento de retina. Foi desenvolvido um processo que permite a conservação de ARN entre os blocos cirúrgicos e laboratoriais. Temos um protocolo padronizado para purificar RNA por ultracentrifugação de cloreto de césio para assegurar que os ARN purificados são adequados para a análise de microarray.

Vídeo: Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Método para Desenvolvimento de Drogas Anti-Câncer Novel utilizando explantes Tumor de espécimes cirúrgicos


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Medicina

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocolo melhorado para microdissecção a laser de ilhotas pancreáticas humanas a partir de espécimes cirúrgicos

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Dois métodos para estabelecer células endometriais Humano estromais primários de espécimes de histerectomia

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

A Passo a Passo Protocolo de Cirurgia sub-retiniana em Coelhos

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

Geração de um humanizado rato Liver Usando células-tronco hepáticas humanas

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

A microscopia intravital da vasculatura Tumor-associado usando avançada Dorsal câmaras de janela dobras cutâneas em ratos transgênicos fluorescentes

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Usando em Vitro viver-pilha de imagem para explorar Chemotherapeutics entregues por nanopartículas de lipídios-baseado

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Recent technological progress has enabled the fabrication of microelectrode arrays that allow recording the activity of multiple individual neurons in the neocortex of the human brain. These arrays have the potential to bring unique insight onto the neuronal correlates of cerebral function in health and disease. Furthermore, the identification and decoding of volitional neuronal activity opens the possibility to establish brain-computer interfaces, and thus might help restore lost neurological functions. The implantation of neocortical microelectrode arrays is an invasive procedure requiring a supra-centimetric craniotomy and the exposure of the cortical surface; thus, the procedure must be performed by an adequately trained neurosurgeon. In order to provide an opportunity for surgical training, we designed a procedure based on a human cadaver model. The use of a formaldehyde-fixed human cadaver bypasses the practical, ethical and financial difficulties of surgical practice on animals (especially non-human primates) while preserving the macroscopic structure of the head, skull, meninges and cerebral surface and allowing realistic, operating room-like positioning and instrumentation. Furthermore, the use of a human cadaver is closer to clinical daily practice than any non-human model. The major drawbacks of the cadaveric simulation are the absence of cerebral pulsation and of blood and cerebrospinal fluid circulation. We suggest that a formaldehyde-fixed human cadaver model is an adequate, practical and cost-effective approach to ensure proper surgical training before implanting microelectrode arrays in the living human neocortex.

We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain.

Treinamento cirúrgico para implantação de microeletrodos Neocortical matrizes usando um modelo de cadáver humano formaldeído-fixo

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...

Diagnosis and Surgical Treatment of Human Brucellar Spondylodiscitis

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in pastoral communities. It is difficult to differentially diagnose and usually leads to irreversible neurologic deficits and spinal deformities. The initial diagnosis of BS is based on clinical findings and radiographic assessments, and the confirmed diagnosis should be established by the isolation of Brucella species from the blood and/or the standard tube agglutination test. Differential diagnosis of multifocal BS from either degenerative disc diseases or tuberculosis is especially highlighted. The surgical approach, either endoscopic or open, is demonstrated in detail, accompanied by radiographic evidence of structural compression or severe instability. Further, the crucial surgical steps, including single-stage transforaminal decompression, debridement, interbody fusion, and internal fixation, are explained. Moreover, perioperative care and postoperative rehabilitation are also addressed. Taken together, this clinical algorithm presents a practical guide that has yielded substantially satisfactory outcomes in the past decades, which can also be introduced for large-scale application to manage human BS, especially in endemic regions.

We describe a clinical algorithm, based on decades of front-line experience of diagnosis and surgical treatment of human Brucellar spondylodiscitis in the largest medical center of the China's Xinjiang Pastoral Area.

Diagnóstico e Tratamento Cirúrgico da Espondilodiscite Humana de Brucellar

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Indução e Análise do Estresse Oxidativo em Células Epiteliais de Pigmento Epitelial Humano Transposon-Transfected Da Bela Adormecida

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...