We thank Dr. Vicky Macias, Angeline Giangreco and Avani Vaishnav for assistance with optimizing this methodology over the years and Yang Zhang and Dr. Jian Ma at the University of Illinois at Urbana-Champaign for the RNA seq analysis. This work was supported by NIH/NCI R01 CA166588-01 (Nonn), American Cancer Society Research Scholar 124264-RSG-13-012-01-CNE (Nonn), NIH/NCI R03 CA172827-01 (Nonn), DOD-CDMPR PRCP Health Disparities Idea Award PC121923 (Nonn) and a Prevent Cancer Foundation grant (Zhou).

Todos os tecidos humanos utilizados para estas experiências foram adquiridas através de um protocolo aprovado Institutional Review Board e / ou isenção na Universidade de Illinois em Chicago. 1. Seção Fresco Congelado próstata para PEN-slide e para Cobrado placa de vidro <ol><li> No dia anterior ou poucas horas antes do corte da amostra, ferramentas limpas (isto é, escova, fórceps, coplins, lâminas, lâminas PEN-emoldurados (se já não estiver RNase livre), um marcador ETOH seguro e um lápis) e no interior de um RT criostato com uma solução de RNase-descontaminação usando um frasco de spray e LAB-toalhetes. <ol><li> Permita que a solução de descontaminação RNase-a descansar por 5 minutos antes de enxugar, enxaguar com DEPCH 2 0 para remover sobra solução e uma lavagem final com 70% EtOH (preparado com DEPCH 2 0) RNase-descontaminação. Nota: É essencial para manter um espaço de trabalho livre de RNase. Todos os coplins, ferramentas, meio de montagem congelados, lâminas e slides utilizados devem ser usados ​​apenas para RNase de trabalho livree nunca usado em um ambiente livre não-RNase padrão. Todos os instrumentos têm de ser tratada com uma solução de descontaminação de RNase antes de cada experiência. </li></ol></li><li> Cryostat legal a -24 ° C. </li><li> Coloque os seguintes instrumentos RNase gratuitas limpos dentro cryostat esfriar por pelo menos 30 min antes do corte: slide com etanol a 100%, as ferramentas, as pequenas cassetes de tecido congelado e chuck montagem cheio de coplin. </li><li> Recuperar a partir de tecido da próstata congelado cryostorage e lugar em um balde espuma seca-cheia de gelo para o transporte. Colocar em criostato de tecido equilibrar à criostato temperatura durante pelo menos 30 min. Nota: tecidos da próstata humanos para a captura de laser de micro-dissecção pode ser fresco congelado ou formalina-fixados em parafina (FFPE). As limitações de tempo e preparação da lâmina são ligeiramente diferentes entre fontes de tecido. Aqui nós descrevemos os passos para cortes congelados. </li><li> Trabalhar com um tecido de cada vez, e esguichar meio de montagem congelado no bottom da cassete e rapidamente montar o tecido prostático congelado para o meio de montagem congelado utilizando a pinça refrigerados. </li><li> Coloque a gaveta com o tecido em resfriador em criostato durante 5 min para definir meio de montagem congelado. </li><li> Tutuca uma pequena quantidade de meio de montagem congelada para o chuck e pressione cuidadosamente tecido montado lado de baixo para cima do meio de montagem congelada. Deixe definido no criostato durante 5 min para permitir que o meio de montagem congelados para solidificar completamente. </li><li> Coloque chuck no suporte e aperte. </li><li> Trave a manivela na posição de bloqueio e coloque uma lâmina nova para o criostato. Para terminais moleculares a jusante que incluem um passo de amplificação (ou seja, qPCR), é aconselhável a utilização de uma lâmina nova para cada paciente para evitar a contaminação (ou mover a lâmina através de utilização da área da lâmina para a amostra seguinte). </li><li> Destravar a alça e cuidadosamente avançar o tecido à mão ou com um pedal para uniformizar e expor o tecido com 50 μsecções M até a exposição do tecido desejado seja alcançado. </li><li> Ajuste cryostat a 5 um, cortar uma ou duas seções e colocá-los em cobrados lâmina de vidro para hematoxilina e eosina (H &amp; E) coloração (ver Passo 2). </li><li> Colocar imediatamente a deslizar para o etanol frio a 100% durante 2 min. </li><li> Remover a lâmina de vidro carregada a partir de etanol e deixar secar à RT, antes de prosseguir para a coloração com H &amp; E (Passo 2). </li><li> Coloque RT PEN-quadro de slide em um torcedor do quadro. </li><li> Ajuste cryostat 10 seções um e lugar para os slides quadro RT PEN. Um a quatro secções pode ser colocado em cada corrediça, dependendo do tamanho do tecido. </li><li> Mergulhar imediatamente a deslizar para o etanol frio a 100% durante 2 min. </li><li> Retirar as lâminas PEN-quadro a partir de etanol e armazenar o slide em uma caixa de slide em uma caixa de secador em um freezer -80 ° C até que esteja pronto para LCM. Três dias é o tempo máximo de armazenamento para recuperação de RNA ideal. </li><li> Prossiga com a Etapa 2 para apenas o vidro cobrado deslizoue. Prossiga com a Etapa 3 para PEN-quadro de slide. </li></ol> 2. Hematoxilina e Eosina-Y (H &amp; E) da mancha por histológica Mark-up Nota: Este caso para o tecido sobre a lâmina de vidro carregada e não o slide PEN-quadro para LCM. <ol><li> Hidratar as secções sobre lâminas de vidro carregada mergulhando a lâmina por meio de etanol graduada como segue: mergulha o slide duas vezes em etanol a 100% durante 3 min, duas vezes em etanol a 95% durante 3 min, uma vez em etanol a 70% durante 3 min e dois vezes em H2O destilada durante 3 min. <ol><li> Preparar a solução de álcool de ácido: 99 ml de etanol a 70% + 1 ml de 1% de HCl e solução de bicarbonato de sódio a 0,1%: 0,1 g de bicarbonato de sódio em 100 ml de H2O destilada </li></ol></li><li> Mergulhe as lâminas em hematoxilina Gill II (0,4%) por 5 min. </li><li> Lavar o excesso de corante em constante funcionamento torneira H2O por 3 min. </li><li> Mergulhe o slide 10 vezes em álcool ácido </li><li> Lave o slide no corrente da torneira H2O por 3 min. </li><li> Mergulhe o slidenuma solução de bicarbonato de sódio a 0,1% durante 30-60 seg para transformar a cor púrpura para azul. </li><li> Lave o slide no corrente da torneira H2O por 3 min. </li><li> Lavar as lâminas em etanol a 95%, com 10 mergulhos. </li><li> Mergulhe o slide no Eosina-Y 2-3 vezes 15 seg total. </li><li> Desidratar o slide através de etanol classificados da seguinte forma: mergulhe a lâmina duas vezes em etanol a 95%, duas vezes em etanol a 100%, e duas vezes em xileno (certifique-se o tecido parece claro o que indica desidratação completa). </li><li> Montar o slide com não aquoso difícil montagem médio e tampa de deslizamento permanente. </li><li> Deixe que a corrediça seco durante 30 min. </li><li> Digitalização de slides com qualquer scanner de slides todo e imprimir uma imagem grande, de alta resolução para 8 &quot;x 11&quot; papel. </li><li> Esboço áreas de interesse (normalmente feito por um patologista certificado bordo) com um marcador. Essa marcação é o guia para o procedimento LCM. </li></ol> 3. Azul de Toluidina coloração das lâminas PEN-quadro Nota: Thestá é feito o mesmo dia como o LCM. Use DEPCH 2 O de água para todas as soluções. Conclua todas as etapas em área livre de RNase. Todos os coplins devem ser limpas com uma solução de RNase-descontaminação. <ol><li> O dia de LCM remover as lâminas PEN-quadro de -80 ° C e hidrato de congelador por lâminas inclinadas em declive em EtOH a 90% durante 2 minutos, em seguida, 75% de EtOH durante 2 min. <ol><li> Prepare 0,5% azul de toluidina por dissolução em água biologia molecular grau em seguida, filtrar através de um filtro de esterilizar e armazenar 0,2 m à temperatura ambiente. Esta solução pode ser reutilizada durante até 6 meses. Nota: É altamente aconselhável para preparar a solução antes que a filtração pode levar algumas horas. </li></ol></li><li> Manchar o tecido da próstata mergulhando suavemente slides em 0,5% azul de toluidina para 5-30 seg. Destain o tecido desliza por lavagem 2 vezes em DEPCH 2 O durante 15 segundos seguido por 75% de EtOH durante 30 segundos a 3 minutos. Nota: mancha e destain tempos podem ser ajustados para assegurar uma boa coloração. Próstata tende a sobre a mancha. </li><li&gt; Transferir as lâminas PEN-quadro para recipiente seco RNAase-livre no gelo. </li></ol> Captura Laser-4. Micro-dissecção (LCM) <ol><li> Imediatamente antes de LCM, a corrediça seco sobre uma lâmina quente a 42 ° C durante 15 min. Dry apenas o slide que será processado e armazenar o resto em gelo até estar pronto para eles. </li><li> Ligue o computador e LCM, por esta ordem, o poder do microscópio, a chave de laser, o interruptor do laser e, em seguida, o computador. O lançamento do software Laser Microdissection. </li><li> Use o software para controlar o microscópio para carregar os slides e tubos de coleta. Clique em &quot;suporte de amostras de descarga&quot;, carregue o slide com as seções no suporte de slides do microscópio com o tecido voltado para baixo e insira o suporte de slides na ranhura apropriada no microscópio, em seguida, clique em &quot;continuar&quot;. <ol><li> Clique em &quot;tubos de coleta de descarregamento&quot;, rótulo e carregar 0,5 ml tubos no suporte do tubo e inseri-lo no slot apropriado no instrumento. Uma veztampas estão garantidos, adicionar 35 ul de tampão de lise para as tampas de coleta e tentar evitar bolhas. Em seguida, clique em &quot;OK&quot;. Nota: Se a evaporação é um problema, dilui-se o tampão da seguinte forma: 25 ul de tampão de lise com 10 ul depcH2O. </li></ol></li><li> No software, seleccionar a posição em que a corrediça tem sido colocada no suporte da lâmina. Selecione a tampa de cobrança para recolher a amostra LCM. </li><li> Visualize e focar o slide através do computador em 10X. Use o software para calibrar o laser, selecionando &quot;Laser&quot; e depois &quot;calibrar&quot;. Ajustar a potência, a abertura ea velocidade do laser, escolhendo por &quot;laser&quot;, em seguida, &quot;controlo&quot; para permitir que o feixe de laser para cortar a área seleccionada completa e eficiente. Repetir até que os ajustes cortes a laser completamente através do tecido. </li><li> Usando o patologista mark-up 8 &quot;x 11&quot; como um guia, use a ferramenta de &quot;chamar a forma&quot; de circunscrever as áreas desejadas de Epítetoslium na vista e evitar a coleta de qualquer estroma. Nota: Várias áreas podem ser selecionadas dentro de um ponto de vista, mas não se movem para outra vista sem primeiro corte clicando a ferramenta &quot;cortar forma&quot;. <ol><li> Se uma área não fica completamente cortada pelo laser de usar a ferramenta &quot;mover e cortar&quot; para passar por isso manualmente. Continue a mover o objetivo de novos pontos de vista e repita o corte a laser até slide é completamente dissecado ou a quantidade desejada de tecido foi coletada (tipicamente 100 glândulas benignas produzir 150-500 ng de RNA). (ver o exemplo na Figura 1A). </li></ol></li><li> Remova cuidadosamente os tubos de suporte à recolha e fechar as tampas, lembrando do tampão de lise e tecido nas calotas. Resumidamente, centrifugar durante 30 segundos para recolher o líquido e o tecido na parte inferior do tubo. Incubar as amostras a 42 ° C durante 30 min e armazenar a -80 ° C até estar pronto para o isolamento de ARN. </li></ol> 5. Isolamento de ARN total com um FilKit baseada em ter e Análise de Qualidade <ol><li> Descongelar os tubos em gelo e levar o volume da solução para 100 uL. </li><li> Pré-molhou a micro filtro de um kit de isolamento de ARN através da aplicação de 30 ul ofLysis Solução para o centro do filtro e deixe de molho whileperforming os dois passos seguintes (pelo menos, 5 min). </li><li> Adicionar 3 ml de solução de LCM Aditivo (fornecido no kit) ao lisado e misturar em vortex durante 5 segundos. Centrifugar durante 30 segundos para recolher o fluido na parte inferior do tubo. </li><li> Centrifugar o filtro pré-humedecido para ~ 30 segundos na velocidade superior para remover o líquido. </li><li> Adicionar 1,25 volumes (neste caso 129 ul) de etanol a 100% para a mistura de lisado, suavemente vórtice ou pipeta cima e para baixo. ** Este método irá recuperar as duas espécies de RNA grandes e pequenos. </li><li> Carregar a amostra sobre o micro-filtro pré-humedecido, e centrifuga-se a 10.000 xg durante 1 minuto para se ligarem a ARN ao filtro. Em seguida, lave o filtro micro com 180 ul de &quot;solução de lavagem 1, R21; centrifugar a 10000 xg durante 1 min. Nota: Deste ponto em diante todas as centrifugações deve ser feito a 13.000 xg, ou velocidade máxima. </li><li> Lavar duas vezes com filtrar 180 ul de &quot;soluções de lavagem 2 e 3&quot;, respectivamente, conforme indicado pelo protocolo do kit. Centrifugar durante 30 segundos, em seguida, descartar o fluxo através de, e girar o filtro durante 1 min para remover o líquido residual e para secar o filtro. </li><li> De transferência do filtro para um novo tubo de recolha. </li><li> Solução quente de eluição a -95 ° C (fornecido com o kit). </li><li> Aplicar 10 ul de solução de eluição pré-aquecido para o centro do filtro, fechar a tampa e armazenar durante 5 min à TA. Em seguida centrifugar durante 1 min para eluir o ARN, e repetir este passo de cada vez, para se obter ~ 20 ul de ARN. </li><li> Realizar o tratamento de DNase I durante 15 minutos a 37 ° C. </li><li> Quantificar o ARN com um espectrof otómetro de absorvência a 260 nm. A relação de densidade óptica no comprimento de onda de 260 nm e 280 nm é utilizado para avaliar a pureza deo RNA (ver Tabela 1) 15. </li></ol> 6. Análise da expressão gênica Nota: A expressão dos genes de espécies de ARN de comprimento (como mRNAs e lncRNAs) é mostrado no passo 6.1 e de espécies de RNA curtas (como microARNs) é mostrado no passo 6.2. Diferentes kits estão disponíveis para RT-qPCR e da quantidade de RNA necessário varia de acordo com kit (tão pouco quanto 10 ng). A análise de sequenciação de ARN é descrito em 6.3. <ol><li> Transcrever reversa (RT) do ARN para ADNc da mistura de 5 mL de 20 ng / ul de amostra de ARN com 5 uL da mistura principal RT (tampão RT, dNTP, iniciadores aleatórios, enzima RT, inibidor de RNase). Incubar a reacção durante 25 min a 25 ° C, em seguida, 120 min a 37 ° C e 5 min a 85 ° C. </li><li> Dilui-se a reacção de RT 1:10 com RNase isenta de água e usar 2,5 mL por 10 jil de reacção qPCR com iniciadores ou conjuntos iniciador / sonda para os genes de interesse. Nota: Para facilitar a análise de RNA parcialmente degradada É aconselhável a utilização de primers for amplicões mais curtos (menos do que 100 pb) 16. (Tabela 2B) <ol><li> Para análise de expressão microARN executar a reacção de RT através da mistura de 5 mL de 2-10 ng / ul de amostra de ARN com 5 uL da mistura principal RT. Nota: cada tecnologia de detecção com base em PCR microARN disponível comercialmente requer a utilização de iniciadores de RT e de propriedade específicos. </li></ol></li><li> Alternativamente ou em adição a RT-PCR, utilizar sequenciação geração seguinte (SEQ ARN-ARN ou de pequena-SEQ) para quantificar diferentes espécies de ARN. Tipicamente, usar 500 ng de ARN para este procedimento (Tabela 3) e levar a cabo o método tal como indicado pelo fabricante e detecção instrumento. </li></ol>

<ol>
	<li>Schauer, I. G., Rowley, D. 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+functional+role+of+reactive+stroma+in+benign+prostatic+hyperplasia.">The functional role of reactive stroma in benign prostatic hyperplasia.</a> Differentiation. 82, 200-210 (2011).</li><li>Peehl, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cell+cultures+as+models+of+prostate+cancer+development.">Primary cell cultures as models of prostate cancer development.</a> Endocrine-related cancer. 12, 19-47 (2005).</li><li>McNeal, J. E., Haillot, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+spread+of+adenocarcinoma+in+the+prostate+as+related+to+cancer+volume.">Patterns of spread of adenocarcinoma in the prostate as related to cancer volume.</a> The Prostate. 49, 48-57 (2001).</li><li>Nonn, L., Vaishnav, A., Gallagher, L., Gann, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+and+micro-RNA+expression+analysis+in+laser-capture+microdissected+prostate+biopsies:+valuable+tool+for+risk+assessment+and+prevention+trials.">mRNA and micro-RNA expression analysis in laser-capture microdissected prostate biopsies: valuable tool for risk assessment and prevention trials.</a> Experimental and molecular pathology. 88, 45-51 (2010).</li><li>Long, Q., 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=Global+transcriptome+analysis+of+formalin-fixed+prostate+cancer+specimens+identifies+biomarkers+of+disease+recurrence.">Global transcriptome analysis of formalin-fixed prostate cancer specimens identifies biomarkers of disease recurrence.</a> Cancer research. 74, 3228-3237 (2014).</li><li>Long, Q., 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=Protein-coding+and+microRNA+biomarkers+of+recurrence+of+prostate+cancer+following+radical+prostatectomy.">Protein-coding and microRNA biomarkers of recurrence of prostate cancer following radical prostatectomy.</a> The American journal of pathology. 179, 46-54 (2011).</li><li>Giangreco, A. 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=Differential+expression+and+regulation+of+vitamin+D+hydroxylases+and+inflammatory+genes+in+prostate+stroma+and+epithelium+by+1,25-dihydroxyvitamin+D+in+men+with+prostate+cancer+and+an+in+vitro.+model.">Differential expression and regulation of vitamin D hydroxylases and inflammatory genes in prostate stroma and epithelium by 1,25-dihydroxyvitamin D in men with prostate cancer and an in vitro. model.</a> The Journal of steroid biochemistry and molecular biology. , (2014).</li><li>Giangreco, A. 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=Tumor+suppressor+microRNAs,+miR-100+and+-125b,+are+regulated+by+1,25-dihydroxyvitamin+D+in+primary+prostate+cells+and+in+patient+tissue.">Tumor suppressor microRNAs, miR-100 and -125b, are regulated by 1,25-dihydroxyvitamin D in primary prostate cells and in patient tissue.</a> Cancer prevention research. 6, 483-494 (2013).</li><li>Mihelich, B. L., 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=miR-183-96-182+cluster+is+overexpressed+in+prostate+tissue+and+regulates+zinc+homeostasis+in+prostate+cells.">miR-183-96-182 cluster is overexpressed in prostate tissue and regulates zinc homeostasis in prostate cells.</a> The Journal of biological chemistry. 286, 44503-44511 (2011).</li><li>Bevilacqua, C., Makhzami, S., Helbling, J. C., Defrenaix, P., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+RNA+integrity+in+a+homogeneous+population+of+mammary+epithelial+cells+isolated+by+Laser+Capture+Microdissection.">Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection.</a> BMC cell biology. 11, 95 (2010).</li><li>Gregg, J. L., Brown, K. E., Mintz, E. M., Piontkivska, H., Fraizer, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gene+expression+in+prostate+cancer+epithelial+and+interstitial+stromal+cells+using+laser+capture+microdissection.">Analysis of gene expression in prostate cancer epithelial and interstitial stromal cells using laser capture microdissection.</a> BMC cancer. 10, 165 (2010).</li><li>Hart, M., 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=Comparative+microRNA+profiling+of+prostate+carcinomas+with+increasing+tumor+stage+by+deep+sequencing.">Comparative microRNA profiling of prostate carcinomas with increasing tumor stage by deep sequencing.</a> Molecular cancer research : MCR. 12, 250-263 (2014).</li><li>Smalheiser, N. R., Lugli, G., Thimmapuram, J., Cook, E. H., Larson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+siRNAs+and+noncoding+RNA-derived+small+RNAs+are+expressed+in+adult+mouse+hippocampus+and+are+up-regulated+in+olfactory+discrimination+training.">Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training.</a> Rna. 17, 166-181 (2011).</li><li>Song, C., 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=Expression+profile+analysis+of+microRNAs+in+prostate+cancer+by+next-generation+sequencing.">Expression profile analysis of microRNAs in prostate cancer by next-generation sequencing.</a> The Prostate. 75, 500-516 (2015).</li><li>Deng, M. Y., Wang, H., Ward, G. B., Beckham, T. R., McKenna, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+six+RNA+extraction+methods+for+the+detection+of+classical+swine+fever+virus+by+real-time+and+conventional+reverse+transcription-PCR.">Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR.</a> Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc. 17, 574-578 (2005).</li><li>Kong, 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=Quantitative+assessment+of+short+amplicons+in+FFPE-derived+long-chain+RNA.">Quantitative assessment of short amplicons in FFPE-derived long-chain RNA.</a> Scientific reports. 4, 7246 (2014).</li></ol>

The authors have nothing to disclose.

A expressão do gene de perfis a partir de amostras humanas pode ser um desafio, não só para a qualidade ou quantidade de tecido disponível, mas também para as várias entidades histológicas presentes numa determinada amostra de tecido. Isto é particularmente difícil na próstata em que os tecidos benignos são em grande parte tecidos estromais e áreas de cancro são desprovidos de estroma. LCM facilita a separação física do estroma prostático e ARN epitélio para uma assinatura mais preciso dos dois tipos d...

A próstata é um tecido heterogéneo composto por epitélio secretor dispostos em ácinos glandular rodeado por estroma fibromuscular composto principalmente de músculo liso 1. O compartimento epitelial é composto por cinco tipos de células diferentes, mas organizados: células basais, células secretoras, células neuroendócrinas, células amplificadoras de trânsito e células-tronco 2. No cancro da próstata (CaP), que surge a partir de células epiteliais luminais, o crescimento do adenocarcinoma provoca um declínio progressivo evidente do compartimento estromal 3. Por estas razões, as amostras de tecido terá diferenças distintas na proporção de estroma e tipo de célula epitelial com base na extensão de CaP. Estas diferenças podem levar a pressupostos enviesada dos dados de expressão de genes adquiridos a partir de tecidos inteiros sem ter em conta a microdissecação de o tipo de célula desejado. Portanto, para eliminar esta polarização é essencial para separar tipos de células antes da extracção de ARN eAnálise da expressão do gene. Macrodissection microdissecação ou pode ser usado para separar fisicamente as áreas epiteliais bem caracterizadas do estroma circundante 4 -6. Macrodissection é tipicamente feito com uma lâmina de barbear sob um microscópio de dissecação e funciona bem para a separação de grandes CaP nódulos de estroma, mas não é capaz de remover a partir de epitélio benigna estroma circundante (ver exemplo de histologia benigna da próstata na Figura 1). Microdissection com um laser (LCM) é significativamente mais trabalho intensivo do que macrodissection, mas pode dissecar com muita precisão epitélio benigno 4. Publicações recentes de nosso laboratório demonstraram que o RNA pode ser extraído com sucesso por LCM de qualquer um (FFPE) biópsias embebidos em parafina fixadas em formalina ou tecido congelado 4,7-9. Os principais desafios na extracção LCM-ARN são: 1) para dissecar com precisão as áreas desejadas do tecido, e 2) para preservar RNA integridade durante o processo de LCM 4,10 e isolamento. ARN isolado a partir de populações de células puras podem ser utilizados para análise da expressão do gene através de vários métodos, incluindo a transcrição reversa-PCR quantitativo (RT-qPCR) 7,8, 11 microarray, e profunda -sequencing 12-14. O objetivo deste protocolo é para isolar RNA total de LCM prostática epitélio de tecido congelado para análise de expressão de genes a jusante.

<table><tbody><tr><td>RNase-AWAY&amp;nbsp;</td><td>MBP</td><td>7005-11</td><td></td></tr><tr><td>PEN-membrane 4.0 mm slides&amp;nbsp;</td><td>Leica</td><td>11600289</td><td></td></tr><tr><td>Glass slides, Superfrost Plus</td><td>FisherBrand</td><td>12-550-15</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>Decon labs.</td><td>2701</td><td></td></tr><tr><td>DEPC (diethyl pyrocarbonate)</td><td>Sigma</td><td>D-5758</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050</td><td></td></tr><tr><td>Coplins (Staining jar)</td><td>IHCWORLD</td><td>M900-12</td><td></td></tr><tr><td>Coplins (Staining rack)</td><td>IHCWORLD</td><td>M905-12</td><td></td></tr><tr><td>Aperio ScanScope</td><td>Aperio(Leica)</td><td>ScanScope&amp;reg; CS</td><td></td></tr><tr><td>Toluidine Blue</td><td>Fluka</td><td>89640-5G</td><td></td></tr><tr><td>Laser Microdissection System</td><td>Leica</td><td>LMD7000</td><td></td></tr><tr><td>0.5 ml Thin-walled Tubes for LCM</td><td>Thermo Scientific</td><td>AB-0350</td><td></td></tr><tr><td>RNAqueous&amp;reg;-Micro Total RNA Isolation Kit</td><td>Ambion</td><td>AM1931</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td>ND-1000</td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Life Technologies</td><td>Q32866</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Applied Biosystems</td><td>4368814</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>Universal cDNA Synthesis Kit II, 8-64 rxns</td><td>Exiqon</td><td>203301</td><td></td></tr><tr><td>TaqMan microRNA RT kit</td><td>Applied Biosystems</td><td>4366597</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>Hematoxylin stain&amp;nbsp;</td><td>Ricca Chemical Company</td><td>3536-16</td><td></td></tr><tr><td>Eosin-Y</td><td>Richard Allan Scientific&amp;nbsp;</td><td>7111</td><td></td></tr></tbody></table>

laser-capture microdissection of human prostatic epithelium for rna analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

Num estudo anterior demonstrou que a utilização de LCM para recolher os tecidos epiteliais e do estroma para comparar perfis de expressão por RT-qPCR de ARNm e microARNs de tecidos da próstata e congelados FFPE a partir do mesmo paciente 4. LCM é demorado, especialmente se grande quantidade de RNA deve ser recolhida para análise de sequenciamento de próxima geração. Portanto, é crucial manter o espaço de trabalho e ferramentas de RNase livre. Recomenda-se para analisar os controlos de qualidade e c...

Watch this Scientific Journal Video about Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis at JoVE.com

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Captura de Laser Microdissection de Próstata Humano Epithelium para Análise de RNA

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

laser-capture-microdissection-human-prostatic-epithelium-for-rna

Research

JoVE Journal

Medicine

13.4K visualizações.  University of Illinois at Chicago. The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Vídeo: Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

Detecção MicroRNA em tumores de próstata por Quantitative PCR em tempo real (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Medicina

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

Sequenciamento de RNA não codificador de tecidos reparados em formalina e exossomos derivados do soro de pacientes com câncer de próstata resistentes à castração

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

Pesquisa do câncer

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

Identificação baseada em Microarray de Individual HERV Loci Expressão: Aplicação a descoberta de biomarcadores em câncer de próstata

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Microdissecção de captura a laser da cartilagem embrionária do rato e osso para análise de expressão gênica

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Biologia do Desenvolvimento

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

As análises de expressão de miRNA no câncer de próstata tecidos clínicos

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

Otimização de captura Laser Microdissection para o isolamento dos gânglios entéricos de tecido humano fresco congelado

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Neurociência

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Isolamento de RNA celular subpopulações específicas Utilizando captura de Laser Microdissection combinada com o rápido immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

Biologia

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

Captação microdissection da malha Trabecular altamente pura de olhos de rato de laser para análise de expressão do Gene

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

Um protocolo da microdissecção da captação do laser que rende o RNA da alta qualidade dos ossos frescos-congelados do rato

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and depth of this technology and extending it to the level of cells within the tissue is needed. Although the use of single nuclear and single cell RNA sequencing has become widespread, the expression signatures of cells obtained from tissue dissociation do not maintain spatial context. Laser microdissection (LMD) based on specific fluorescent markers would allow the isolation of specific structures and cell groups of interest with known localization, thereby enabling the acquisition of spatially-anchored transcriptomic signatures in kidney tissue. We have optimized an LMD methodology, guided by a rapid fluorescence-based stain, to isolate five distinct compartments within the human kidney and conduct subsequent RNA sequencing from valuable human kidney tissue specimens. We also present quality control parameters to enable the assessment of adequacy of the collected specimens. The workflow outlined in this manuscript shows the feasibility of this approach to isolate sub-segmental transcriptomic signatures with high confidence. The methodological approach presented here may also be applied to other tissue types with substitution of relevant antibody markers.

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

Aplicação de microdisseção a laser para descobrir transcrição regional em tecido renal humano

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and ...