Obrigado a Kevin Miyashiro para revestimento e manutenção de culturas de células, ao Dr. Terri Schochet para a prestação de culturas de células para as fotos incluídas neste documento. Além disso, obrigado a Kevin Miyashiro, Peter Dr. Buckley, e Pertiz Tiina para a entrada sobre o procedimento ARNA. Financiamento para este trabalho foi do Instituto Nacional do Envelhecimento, o Instituto Nacional de Saúde Mental e Recursos Humanos fundos Fact Finder a partir da Pensilvânia.

<ol>
	<li>Davis, J. E., Eberwine, J. H., Hinkle, D. A., Marciano, P. G., Meaney, D. F., McIntonsh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+regarding+single-cell+gene+expression+profiling+for+brain+injury.">Methodological considerations regarding single-cell gene expression profiling for brain injury.</a> Neurochemical Research. 29, 1113-1121 (2004).</li><li>Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+molecular+biology.">Single-cell molecular biology.</a> Nature Neuroscience. 4, 1155-1156 (2001).</li><li>Kamme, F., Salunga, R., Yu, J., Tran, D., Zhu, J., Luo, J., Bittner, A., Guo, H., Miller, N., Wan, J., Erlander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+microarray+analysis+in+hippocampus+CA1:+Demonstration+and+Validation+of+Cellular+Heterogeneity.">Single-cell microarray analysis in hippocampus CA1: Demonstration and Validation of Cellular Heterogeneity.</a> The Journal of Neuroscience. 23, 3607-3607 (2003).</li><li>Hinkle, D., Glanzer, J., Sarabi, A., Pajunen, T., Zielinski, J., Belt, B., Miyashiro, K., McIntosh, T., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+neurons+as+experimental+systems+in+molecular+biology.">Single neurons as experimental systems in molecular biology.</a> Progress in Neurobiology. 72, 129-142 (2004).</li><li>Ginsberg, S. D., Elarova, I., Ruben, M., Tan, F., Counts, S. E., Eberwine, J. H., Trojanowski, J. Q., Hemby, S. E., Mufson, E. J., Che, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+gene+expression+analysis:+Implications+for+Neurodegenerative+and+Neuropsychiatric+Disorders.">Single-cell gene expression analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders.</a> Neeurochemical Research. 29, 1053-1064 (2004).</li><li>Eberwine, J., Spencer, K., Miyashirto, K., Mackler, S., Finnell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+DNA+synthesis+in+situ:+methods+and+applications.">Complementary DNA synthesis in situ: methods and applications.</a> Methods Enxymol. 216, 80-100 (1992).</li><li>Eberwine, J., Yeh, H., Miyashiro, K., Cao, Y., Nair, S., Finnell, R., Zettel, M., Coleman, P. <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+single+live+neurons.">Analysis of gene expression in single live neurons.</a> Proc. Natl. Acad. Sci. USA. 89, 3010-3014 (1992).</li><li>Kelz, M. B., Dent, G. W., Therianos, S., Marciano, P. G., McIntosh, T. K., Coleman, P. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+antisense+RNA+amplification+and+microarray+analysis+as+a+tool+for+studying+neurological+degeneration+and+restoration.">Single-cell antisense RNA amplification and microarray analysis as a tool for studying neurological degeneration and restoration.</a> Sci. Aging Knowledge Eviron. 1, re1-re1 (2002).</li><li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+dispersed+cell+culture.">Rat hippocampal neurons in dispersed cell culture.</a> Brain Research. 126, 397-425 (1977).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> P-1000 &#38; P-97 Pipette Cookbook. , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MinElute Handbook For MinElute Reaction Cleanup Kit. , 28-29 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Megascript kit. , 7-8 .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MEGAclear kit. , 4-5 .</li><li>Eberwine, J., Kacharmina, J. E., Andrews, C., Miyashiro, K., McIntosh, T., Becker, K., Barrett, T., Hinkle, D., Dent, G., Marciano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+expression+analysis+of+tissue+sections+and+single+cells.">mRNA expression analysis of tissue sections and single cells.</a> The Journal of Neuroscience. 21, 8310-8314 (2001).</li><li>Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., Eberwine, J. H. <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> Proc. Natl. Acad. Sci. USA. 87, 1663-1667 (1990).</li><li>Miyashiro, K., Dichter, M., Eberwine, J. <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+nature+and+differential+distribution+of+mRNAs+in+hippocampal+neuritis:+Implications+for+neuronal+functioning.">On the nature and differential distribution of mRNAs in hippocampal neuritis: Implications for neuronal functioning.</a> Proc. Natl. Acad. Sci. USA. 91, 10800-10804 (1994).</li><li>Sul, J., Wo, C. K., Zeng, F., Jochems, J., Lee, M. T., Kim, T. K., Peritz, T., Buckley, P., Cappelleri, D. J., Maronski, M., Kim, M., Kumar, V., Meaney, D., Kim, J., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+transfer+produces+a+predictable+cellular+phenotype.">Transcriptional transfer produces a predictable cellular phenotype.</a> Proc. Natl. Acad. Sci. USA. 106, 7624-7629 (2009).</li><li>Sow, F. B., Gallup, J. M., Sacco, R. E., Ackerman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+revisited+as+a+tool+for+transcriptomic+analysis:+Application+of+an+excel-based+qPCR+preparation+software+(PREXCEL-Q).">Laser capture microdissection revisited as a tool for transcriptomic analysis: Application of an excel-based qPCR preparation software (PREXCEL-Q).</a> Int. J. Biomed. Sci. 5, (2010).</li><li>Vandewoestyne, M., Deforce, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+forensic+research:+a+review.">Laser capture microdissection in forensic research: a review.</a> Int J Legal Med. 124, (2010).</li><li>Eberwine, J., Bartfai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptomics+of+hypothalamic+warm+sensitive+neurons+that+control+core+body+temperature+and+fever+response+Signaling+asymmetry+and+an+extension+of+chemical+neuroanatomy.">Single cell transcriptomics of hypothalamic warm sensitive neurons that control core body temperature and fever response Signaling asymmetry and an extension of chemical neuroanatomy.</a> Pharmacol. Ther. , (2010).</li></ol>

Dr. Eberwine é um inventor da patente ARNA que foi licenciado para LBS tecnologias em que Dr. Eberwine é um accionista.

Notas e solução de problemas <ul><li> Você pode querer tomar antes e depois de imagens mostrando que a célula-alvo foi isolado e que a penumbra restante foi deixado intocado. </li><li> Se demasiada solução é entrar na pipeta como você está colhendo, você pode usar 3 vias Luer-Lock e acessórios para o êmbolo da seringa para manter pressão positiva na tubulação. O volume desejado irá variar com o tipo de processamento, mas até 5 mL deve ser bom para a maioria das aplicações. </li></ul> Dicas gerais sobre técnicas da biologia molecular <ul><li> Vortex todos os tubos de ações e spin-las antes de adicionar a uma reação. NUNCA enzimas vortex. </li><li> Misturar bem reações antes de adicionar a enzima para garantir que o buffer está na concentração adequada. </li><li> Manter o estoque de enzimas a -20 ° C, se possível usando um stratacooler. Caso contrário, remova direito antes do uso, manter no gelo, e retornar imediatamente a -20 ° C. </li><li> Sempre fazer misturas de mestre na elaboração de mais de uma reação para reduzir os erros de pipetagem. Calcular o volume necessário com base no número de amostras e adicionar 10-20%. </li><li> Loja RNA a -80 ° C para a degradação retard. RNA é mais estável armazenados em buffer para retardar apurination de RNA que ocorre em condições ácidas. </li></ul> Esquema Geral do Procedimento ARNA Figura 4A ilustra a primeira rodada do processo de ARNA. Na reação de primeira vertente, a parcela de poli-T da T7-oligo primer (dT) seleciona para as espécies de mRNA (retângulo branco longo) ligando-se à cauda poli. Alguns microRNAs são também polyadenylated e será capturado por esse procedimento. Mais importante, porém, os RNAs mais abundante na célula, RNAs ribossomais, não. Este oligo funciona como um primer para transcriptase reversa para sintetizar uma fita complementar de cDNA (retângulo cinza longo) usando o mRNA como um modelo. A porção do T7 T7-oligo primer (dT) incorpora o promotor T7 RNA polimerase no quadro com a seqüência de antisense para o mRNA de partida. Esta é usada mais tarde na reação in vitro de transcrição. Em seguida, o mRNA no híbrido mRNA / DNA criado na etapa anterior é parcialmente hidrolisada por Rnase H criação de RNA &quot;primers&quot; (pequenos retângulos brancos), semelhante aos fragmentos Okazaki criado em síntese lagging strand DNA. DNA Polimerase I utiliza fragmentos de RNA para preparar a síntese de DNA usando o DNA complementar ao mRNA como um modelo. Quando atinge o fragmento de RNA que vem, seus 5 &#39;para 3&#39; atividade nuclease remove o ribonucleotídeos e substitui-los com desoxirribonucleótidos. DNA ligase é adicionado a qualquer ligadura vertentes em que a substituição da fita líder não está completo. Polimerase DNA T4 é adicionada para preencher as áreas onde fragmentos de RNA serviu como primers inicial para Polimerase DNA eu criar uma ponta e dupla fita de cDNA, que depois é purificado antes de realizar a reação IVT. Na reação IVT, T7 RNA polimerase se liga ao promotor T7 incorporados ao cDNA dupla fita e sintetiza moléculas de RNA antisense (long retângulos pretos), utilizando a vertente sentido como um modelo. Isto serve como etapa de amplificação em que milhares de moléculas de RNA antisense são produzidos a partir de cada molécula de cadeia dupla de cDNA (Figura 4A). A segunda rodada, conforme apresentado na Figura 4B, começa com uma reação de transcrição reversa, que é ligeiramente diferente da primeira rodada desde o RNA de partida é antisense (retângulo preto sólido) e não tem o rabo polyadenylated que foi alvo pela T7-oligo (dT) primário no primeiro turno. Portanto, essa reação é preparado com primers aleatórios (pequenos retângulos cinza) e posteriormente o RNA desnaturado. A reação segunda vertente é, então, preparado pela T7-oligo primer (dT), que se liga ao poli-A seqüência na extremidade 3 &#39;do RNA sentido criado na reação de transcrição reversa anteriores. Outra reação IVT é realizada na mesma maneira que no primeiro turno. Esta segunda ronda geralmente é repetido pelo menos uma vez para atingir três rounds de amplificação de uma única célula. Aplicações As técnicas que apresentamos neste artigo pode ser traduzido em um grande número de aplicações. O protocolo de isolamento única célula pode ser modificado para uso em fatias aguda. 14 Embora tecnicamente mais desafiador, os mesmos princípios se aplicam nesta preparação alternativo. Além disso, se o tamanho da pipeta é ligeiramente ajustada, as gravações da fisiologia das células pode ser feita antes da colheita permitindo uma investigação bem controlados dos mecanismos moleculares por trás saídas fisiológicas. Outra pequena modificação é isolar os processos da soma das células. 15 Para esta aplicação, recolher corpos celulares com uma pipeta e depois voltar com uma pipeta nova e recolher 100-300 Dendrit identificadoses ou axônios por tubo de coleta. 16 Uma vez que as células foram colhidas, as comparações de abundância de mRNA e composições podem ser feitas entre diferentes e mesmo dentro da mesma célula populações. Incorporando-biotinilado UTP no ARNA terceira rodada microarray permite a análise para determinar esses abundâncias mRNA relativo. A composição da população mRNA original pode também ser determinada após o procedimento ARNA utilizando seqüenciamento da próxima geração. O ARNA amplificados também podem ser usados ​​para confirmar os estudos de células fenótipo de conversão em que um conjunto completo de mRNAs de um tipo de célula são transfectadas em um tipo de célula diferente, a fim de induzir a transição do fenótipo do tipo de célula última em que do primeiro, um procedimento desenvolvido pelo laboratório e conhecido como TIPeR 17. Esses estudos são particularmente úteis para o estudo de estados de doença e fenótipos celulares e tais estudos estão em andamento no laboratório. RT-PCR ou qPCR pode ser realizada sobre o material amplificado para confirmar a expressão de genes específicos de células. Além disso, as avaliações da eficiência de transfecção ou transdução pode ser feita no nível da célula única. Vantagens e Limitações Como dito no resumo, o isolamento de células individuais para análise elimina os efeitos média de visto com a análise de populações de células heterogêneas. Estes efeitos média deturpar abundâncias mRNA em uma única célula por excesso de transcrições representando abundante e média para fora e evitar a detecção de muitos de baixa abundância transcrições. Citometria de fluxo pode ser usado para classificar as células individuais, mas este método requer o conhecimento de marcadores celulares específicos e equipamentos caros. 18 microdissecção de captura a laser ou com sistemas de laser UV ou IR microdissecção de captura permitem única célula e até mesmo a captura subcelulares, mas requer células de interesse para estar localizados na superfície de seções muito finas. 19 Semelhante a citometria de fluxo, laser microdissecção de captura também exige equipamentos caros. Um dos principais benefícios da técnica de coleta de eletrodo baseado descrito acima é que valiosos dados eletrofisiológicos pode ser obtido a partir da célula de interesse antes da colheita, permitindo a análise funcional e transcriptoma a ser realizada na mesma célula. 20 A desvantagem da nossa técnica é que exige experiência com micromanipuladores. Investigadores familiarizados com micromanipuladores encontrará esta técnica muito intuitiva, no entanto, os indivíduos com tal experiência será necessário tornar-se confortável com os movimentos bem necessário. Inerentes a qualquer técnica de amplificação é a amplificação preferencial de certas transcrições com base no tamanho e na composição de nucleotídeos. 4,6,7 reação em cadeia da polimerase (PCR), tais como técnicas baseadas transcrição reversa-reação em cadeia da polimerase (RT-PCR) e amplificação rápida de cDNA termina resultado (RACE) na amplificação exponencial de transcrições, enquanto que o procedimento de amplificação ARNA resultados na amplificação linear. Assim, uma das principais vantagens do procedimento ARNA reside na sua capacidade de preservar melhor a abundância relativa de transcritos mRNA por apenas linearmente ampliando quaisquer erros ou desvios que ocorrem no processo de amplificação em oposição a amplificar exponencialmente disse erros e distorções. O procedimento ARNA juntamente com a análise de microarray permite a comparação da abundância de mRNA entre as células individuais da morfologia semelhantes ou diferentes, tratada ou não. Além disso, material amplificado podem ser enviadas para o seqüenciamento da próxima geração. 5,8 No entanto, o cuidado deve ser tomado em tais análises, pois, em contraste com o uso de primers aleatórios para o procedimento, o procedimento de oligo-dT preparado descrito acima preconceitos amplificação do 3 &#39;extremidades do mRNA e geralmente resulta em redução ligeira de material amplificado subseqüente a cada rodada. Cuidados devem ser tomados na análise dos resultados microarray com métodos padrão como algumas chamadas falsas ausentes podem surgir a partir ligeiramente encurtado material amplificado. Além disso, os resultados enquanto o seqüenciamento vai realmente proporcionar a plena 5 &#39;seqüência da maioria dos mRNA original, a 5&#39; seqüências de alguns mRNA pode ser desperdiçada. Por estas razões, o número de rodadas de amplificações devem ser limitados.

A correction was made to <a href="http://http://www.jove.com/video/2634/transcriptome-analysis-of-single-cells">Transcriptome Analysis of Single Cells</a>. There were errors in the volumes used in the aRNA Amplification section.

Erratum: Transcriptome Analysis of Single Cells

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Spiegelgas coverslips</td>
<td>&nbsp;</td>
<td>Carolina Biological</td>
<td>63-3029</td>
<td>&nbsp;</td>
<tr>
<td>Nunc 35x10 mm culture dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-565-90</td>
<td>&nbsp;</td>
<tr>
<td>Water for cell culture</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-724Q</td>
<td>For making solutions used for cell culture and rinsing coverslips</td>
</tr>
<tr>
<td>Poly-D-Lysine MW70-150K</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>6407</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, ultrapure</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354239</td>
<td>&nbsp;</td>
<tr>
<td>Boric acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0252</td>
<td>&nbsp;</td>
<tr>
<td>MEM with Earle&#x2019;s salts and glutamax</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41090-101</td>
<td>For plating cells</td>
</tr>
<tr>
<td>D-glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8769</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050</td>
<td>&nbsp;</td>
<tr>
<td>Cytosine beta-D-arabinofuranoside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1768</td>
<td>&nbsp;</td>
<tr>
<td>MEM with Earle&#x2019;s salts and L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11095-098</td>
<td>For growing cells</td>
</tr>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2256</td>
<td>&nbsp;</td>
<tr>
<td>B27 serum-free supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass capillary tubes (1.5mm O.D, 100mm length)</td>
<td>&nbsp;</td>
<td>Kimble Chase</td>
<td>34500 99</td>
<td>Any borosilicate glass pipette will work as long as the proper bore size is attained. </td>
</tr>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14175</td>
<td>Any solution will work as long as the components won&#x2019;t interfere with any future processing (i.e. no Ca2+ or Mg2+)</td>
</tr>
<tr>
<td>1ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309628</td>
<td>&nbsp;</td>
<tr>
<td>Needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>Gauge depends on the diameter of the pipettes</td>
</tr>
<tr>
<td>dNTP mix</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>28-4065-51</td>
<td>&nbsp;</td>
<tr>
<td>dt-T7 oligo</td>
<td>&nbsp;</td>
<td>Custom</td>
<td>Midland Certified</td>
<td>&nbsp;</td>
<tr>
<td>Second strand buffer (5x)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Y01129</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Supplied with second strand buffer</td>
</tr>
<tr>
<td>E.coli DNA Ligase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100002324</td>
<td>&nbsp;</td>
<tr>
<td>DNA polymerase I</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100004926</td>
<td>&nbsp;</td>
<tr>
<td>Rnase H</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18021-071</td>
<td>&nbsp;</td>
<tr>
<td>Megascript T7 kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1334</td>
<td>&nbsp;</td>
<tr>
<td>Random primers</td>
<td>&nbsp;</td>
<td>BMB</td>
<td>11034731001</td>
<td>&nbsp;</td>
<tr>
<td>Superscript III Reverse Transcriptase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>56575</td>
<td>in kit (18080-044) comes with first strand buffer (Y02321) and DTT</td>
</tr>
<tr>
<td>MEGAclear Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>1908</td>
<td>&nbsp;</td>
<tr>
<td>MinElute Reaction Cleanup Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28206</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA polymerase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100004994</td>
<td>&nbsp;</td>
<tr>
<td>Rnasin</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>N251B</td>
<td>&nbsp;</td>
<tr>
<td>Illumina TotalPrep RNA Amplification Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AMIL1791</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown micropipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-87</td>
<td>Sutter has many other models, many are discussed in the cookbook</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Many other micromanipulators will work such as the newer Eppendorf models</td>
</tr>
<tr>
<td>Pipette Holder</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>MP-S15A</td>
<td>Will vary with micromanipulator and pipette O.D.</td>
</tr>
<tr>
<td>Bioanalyzer RNA 6000 Nano Kit</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>5067-1511</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transcriptome analysis of single cells

Muitas técnicas de análise de expressão gênica confiar no material isolado de populações heterogêneas de células de homogeneizados de tecidos ou células em cultura. 1,2,3 No caso do cérebro, regiões, tais como o hipocampo contém um arranjo complexo de diferentes tipos de células, cada uma com distintos perfis de mRNA. A capacidade de colheita de células individuais permite uma maior investigação detalhada sobre as diferenças molecular entre e dentro de populações de células. Nós descrevemos um método simples e rápido para a colheita de células para posterior processamento. Pipetas muitas vezes utilizados em eletrofisiologia são utilizados para isolar (por meio de aspiração) uma célula de interesse e convenientemente depositá-lo em um tubo Eppendorf para posterior processamento com qualquer número de técnicas de biologia molecular. Nosso protocolo pode ser modificada para a safra de dendrites de cultura de células ou até células individuais de fatias aguda. Descrevemos também o método de amplificação ARNA como uma das principais aplicações a jusante de isolamentos única célula. Este método foi desenvolvido anteriormente por nosso laboratório como uma alternativa a outras técnicas de expressão de genes de análise, como transcrição reversa em tempo real ou reação em cadeia da polimerase (PCR). 4,5,6,7,8 Esta técnica prevê ampliação linear do RNA polyadenylated começando com femtogramas só de material, resultando em quantidades micrograma de RNA antisense. O material amplificado linearmente fornece uma estimativa mais precisa do que a amplificação exponencial da abundância relativa dos componentes do transcriptoma da célula isolada. O procedimento básico consiste em dois rounds de amplificação. Resumidamente, a T7 RNA polimerase promotor local é incorporada dupla cDNA encalhado criado a partir de transcritos no mRNA. Uma noite de transcrição in vitro de reação (FPI) é então feita em que T7 RNA polimerase produz muitas transcrições antisense do cDNA dupla fita. A segunda rodada repete este processo, mas com algumas diferenças técnicas desde a matéria-prima é antisense RNA. É padrão para repetir o segundo turno, resultando em três rounds de amplificação. Muitas vezes, a terceira rodada in vitro a reação de transcrição é realizada utilizando-nucleosídeos trifosfatos biotinilado para que o RNA antisense produzido pode ser cruzados e detectado em um microarray. 7,8

Watch this Scientific Journal Video about Transcriptome Analysis of Single Cells at JoVE.com

Transcriptome Analysis of Single Cells

Neste artigo descrevemos um método simples para a colheita de células isoladas de ratos culturas primárias neuronais e análise de transcriptoma subsequentes usando a amplificação ARNA. Esta abordagem é generalizável a qualquer tipo de célula.

Análise do transcriptoma de uma única célula

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Research

JoVE Journal

Neuroscience

29.7K visualizações.  University of Pennsylvania. Neste artigo descrevemos um método simples para a colheita de células isoladas de ratos culturas primárias neuronais e análise de transcriptoma subsequentes usando a amplificação ARNA. Esta abordagem é generalizável a qualquer tipo de célula.

Vídeo: Transcriptome Analysis of Single Cells

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Em células vivas, imagens de células precursoras de órgãos sensoriais na Intact Drosophila As pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Neurociência

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequencial de foto-branqueamento de delinear células de Schwann individuais na junção neuromuscular

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Marcação de células individuais no Sistema Nervoso Central de<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Laser Microdissecção Captura de Populações enriquecida de neurônios ou neurônios individuais para análise de expressão gênica após a lesão cerebral traumática

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

Olhando para Cérebro polissomos com Microscopia de Força Atômica

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

Single-cell RNA-Seq de subgrupos definidos de células de gânglio retiniano

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Análise de células do sistema imunológico em único nervos ciático e gânglio da raiz Dorsal de um único rato usando citometria de fluxo

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Isolamento dos núcleos de adultos da medula espinhal para a sequenciação do ARN de Single-núcleo maciçamente paralelo

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Crônica implantação de matriz Electrocorticographic todo-cortical no sagui comum

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

Análise da localização de snRNA Spliceosomal em células HeLa humanas usando microinjeção

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...