The authors would like to thank members of the Yi laboratory especially Zhaojie Zhang for fruitful discussions regarding linker design and ligation efficiencies, as well as the American Cancer Society for supporting this work through a postdoctoral fellowship (#125209) to J.E.L. Research reported in this publication was also supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR059697 (to R.Y.) and a research grant from the Linda Crnic Institute for Down Syndrome. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NOTA: É crítico para manter condições isentas de RNase durante todo o procedimento. 1. adenilação de 3 &#39;Linker <ol><li> Diluir oligonucleótido de ADN concebidos para reacções 3 &#39;de ligação para 100 uM em meio livre de nuclease de H 2 O. NOTA: O oligonucleótido deve ter um &#39;grupo fosfato, um dinucleótido randomizados no terminal 5&#39; 5 fim, e um &quot;3 didesoxicitosina. A 5 &#39;grupo fosfato é um requisito da enzima Mth para eficiente 5&#39; 10 adenilação. O fosfato pode ser adicionado ao pré-tratamento do oligonucleótido com um polinucleótido-quinase, ou encomendado com a modificação directa. O dinucleótido randomizado na extremidade 5 &#39;é importante para minimizar a preferência ligases de ARN que exibem determinados para-extremidade final juntando reacções sobre os outros seis sequência. A 3 &#39;didesoxicitosina coloca um elemento de bloqueio na extremidade 3&#39; do oligonucleótido to inibir a formação de concatâmeros ligante-ligante durante os passos de ligação subsequentes e também serve para bloquear &#39;extremidade da molécula de miARN-Ligante formada na extremidade de 3&#39; da reacção de ligação 3 11. A sequência de oligonucleótido ligante escolhido para o método de miR-Seq imparcial é como se segue: 5&#39;PO 4 NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3 &#39;. </li><li> Montar o seguinte reacção adenilação: 200 pmol 3 &#39;oligonucleótido ligante, 4 ul 10x 5&#39; tampão de adenilação de ADN, ATP 1 mM de 4 ul, 4 ul de RNA-ligase Mth, livre de nuclease H 2 O para um volume final de 40 ul. </li><li> Incubar reacção a 65 ° C durante 1 hora. Terminar a reacção por aquecimento a 85 ° C durante 5 min. </li><li> Precipitar ligante adenilada pela adição de 2,5 volumes de etanol a 100% e 1/3 volume de 10 M de acetato de amónio para remover o ATP residual e para evitar imprevistos em ligaduras reacções futuras. <ol><li> Resumidamente, misture o alcohol, sal, oligo e solução até à homogeneidade, girando à velocidade máxima numa microcentrífuga (~ 15.000 xg) a 4 ° C durante 20 min. Lavou-se a pelota em etanol a 70%, ar seco, e ressuspender em um volume apropriado de livre de nuclease H 2 O para dar 50-100 uM de oligo adenilada. </li></ol></li><li> Confirme adenilação executando um gel de poliacrilamida 18%. <ol><li> Prepare o gel por mistura do seguinte em conjunto: 6,3 g de ureia, 6,75 ml de 40% de acrilamida, e 1,5 ml de 10x TBE. Adicionar H2O a 15 ml. </li><li> Misturar até que a ureia é completamente dissolvido e adicionar 150 ul 10% (w / v) de persulfato de amónio (APS) e 15 ul tetramethlyethylenediamine (TEMED). </li><li> Depois gel definiu, pré-corrida por 30 minutos a 24 V / cm de largura com gel TBE 1x tampão de corrida à temperatura ambiente. Depois de 30 minutos é de até poços lave com tampão de corrida. </li><li> Misturar 5 pmol de oligonucleótidos adenilada e com igual volume de tampão de carga desnaturante ARN 2x (95% (v / v) de formamida,0,02% (w / v) de SDS, 0,02% de azul de bromofenol (w / v), 0,01% (w / v), xileno cianol, EDTA 1,0 mM), o calor rapidamente para 75 ° C, e SNAP arrefecida em gelo. Dispensar amostra inteira dentro do poço do gel. Também incluem uma quantidade idêntica de controle não-adenilada e baixos padrões de tamanho peso molecular. </li><li> Submeter o gel a 24 V / cm até o azul de bromofenol é ¾ do caminho para o fundo do gel. Desmonte aparelho, gel pós-mancha com uma solução de SYBR-gold 1x em 1x TBE e visualizar na caixa UV-transilluminator (Figura 2). NOTA: Gel purificar o oligo adenilada de um modo semelhante ao descrito acima, se &quot;nenhum ARN de entrada&quot; controles estão resultando numa elevada quantidade de produto de PCR, em comparação com amostras com níveis de ARN de entrada. </li></ol></li></ol> 2. Ligador Ligadura para 3 &#39;Fim da miARN <ol><li> 3 &#39;Ligadura <ol><li> Montar os seguintes componentes em gelo, na ordem indicada: 1,0 ul de 50% (w / v) de PEG 8000, 0,5 &amp;# 956; l 10x tampão de ligase de RNA (500 mM Tris-HCl pH 7,5, 100 mM MgCl2, 10 mM DTT), 1,0 ul adenilada 3 &#39;Ligante (100 uM), 0,5 ul de inibidor de RNase, 0,5-8,0 ug de RNA total, 0,5 ul de RNA-ligase de T4 2, livre de nuclease H 2 O para um volume final de 5 mL. </li><li> Misturar por pipetagem a reacção como a solução é demasiado viscoso para vórtice, devido à alta concentração de PEG 8000. Uma vez cuidadosamente misturado, coloque a reacção num termociclador com uma tampa aquecida. Definir o bloco a 16 ° C, e incubar durante 4 horas. NOTA: A reacção pode ser dimensionado para um volume final de 20 ul, sem qualquer perda na eficiência da ligação. Reagir durante 4 horas à temperatura ambiente ou durante a noite a 4 ° C, para se obter eficiências de ligação essencialmente idênticos. </li></ol></li><li> Gel Purificação <ol><li> Após 3 &#39;ligadura, misturar a mistura reaccional com um volume igual de 2x tampão de carga de ARN. Aquece-se a 70 ° C durante 5 min, e encaixe fria em gelo. </li&gt; <li> Correr a amostra num gel de poliacrilamida a 15% para a purificação PAGE. <ol><li> Prepare-se um gel de poliacrilamida a 15% contendo ureia 7 M, de um modo semelhante ao descrito anteriormente no passo 1.5. </li><li> Pré-correr o gel a 24 V / cm de largura do gel para, pelo menos, 30 min. Desligue a alimentação, lavar poços, amostra de carga e gel corrida em mesma tensão como pré-corrida. </li><li> Coloque a amostra e execute a página até o azul de bromofenol acaba de sair o gel. O produto desejado migrará perto do cianol xileno. </li></ol></li></ol></li><li> Coloração, visualização e Excisão <ol><li> Desmonte aparelho de gel e coloque gel na bandeja limpa com 1x tampão de corrida (TBE), 1x SYBR-ouro e pedra para 15 min. </li><li> Retire o gel da coloração prato e visualizar na caixa transiluminador UV coberta com filme plástico limpo. O produto de ligação deve ser cerca de 45 nucleótidos de comprimento. </li><li> Impostos especiais de consumo da região que contém o produto da ligação com uma razo limpor lâmina e coloque em 0,5 ml tubo micro-centrífuga. </li></ol></li><li> Isolamento produto e precipitação <ol><li> Pierce fundo do tubo 0,5 ml de microcentrífuga com uma agulha de 30 G e colocar o tubo perfurado em um segundo limpo, baixa retenção, 1,5 ml tubo de microcentrífuga. Centrifugar durante 5 min a 15.000 x g ~. </li><li> Visualmente confirmar que toda a fatia de gel foi movido através do furo no tubo interior. Se necessário, use uma ponta de pipeta limpa para empurrar alguns fragmentos de gel mais perto do buraco. </li><li> Descartar o tubo vazio de 0,5 ml e adicionar 400 ul de tampão de coluna de alto teor salino (HSCB NaCl 400 mM, 25 mM Tris-HCl pH 7,5, SDS a 0,1%) para a pasta de acrilamida. Tubo (s) de rocha, a 4 ° C durante a noite. </li><li> Transferência de lodo a uma coluna de rotação de acetato de celulose de 0,22 micron, com uma pipeta de ponta de todo o furo. Centrifugar durante 3 minutos à temperatura ambiente em ~ 15.000 xg para recolher todo o líquido para longe da matriz de gel de acrilamida. </li><li> Transferir o líquido para um, de baixa retenção limpo, tubo de 1,5 ml contering 1 ml de 20 mg / ml de glicogênio. Adicionar um volume igual de isopropanol e 1/10 de volume de acetato de sódio. Misturar a solução por inversão do tubo várias vezes e precipitar no freezer -80 ° C durante 20 min. </li><li> Centrifugar a plena velocidade a 4 ° C durante 20 min para sedimentar o ácido nucleico. Remover o sobrenadante e lava-se em etanol a 70%. Ar sedimento seco durante 10 min à temperatura ambiente e seguir directamente a 5 &#39;passo de ligação. </li></ol></li></ol> 3. Ligador Ligadura para 5 &#39;Fim da miARN-3&#39; Ligador híbrido <ol><li> Para o ácido nucleico sedimentadas, adicionar os seguintes componentes: 2 uL de PEG 8000 (50% w / v), 0,5 ul de RNA-ligase 10x Tampão, 0,5 ul de ATP (10 mM), 0,5 ul de RNA-NN oligo (100 uM), e H 2 O para um volume final de 4 mL. NOTA: A composição do &#39;oligo NN-ARN &quot;é como se segue 5&#39;-didesoxi-TCUACAGUCCGACGAUCNN3 invertido&#39; e foi purificado por HPLC pelo fabricante. </li><li> Misturaresta amostra por pipetagem cima e para baixo repetidamente. Fazer isso há algum tempo (até 5 minutos), para assegurar que o sedimento foi completamente re-solubilizado. Se a re-solubilização é problemático, aquecer a amostra brevemente (3-5 min) a 37 ° C e retomar pipetagem para misturar. </li><li> Uma vez que o sedimento é de volta em solução, transferir o conteúdo para um tubo limpo de PCR contendo 1 ml de uma mistura 1: 1 de inibidor de RNase e T4 RNA ligase 1 (5 unidades). Novamente misture por pipetagem cima e para baixo. </li><li> Incubar a reacção num termociclador a 37 ° C durante 4 h. Prosseguir imediatamente para a síntese de cDNA. </li></ol> 4. Transcrição Reversa / cDNA Synthesis <ol><li> Adicionar os seguintes componentes para a 5 &#39;da reacção de ligação: 2 ul de tampão 5x RT, 0,75 ul de DTT 100 mM, 1 ul Iniciador RT Ilumina-GCCTTGGCACCCGAGAATTCCA 5&#39;-3&#39; (1 uM), e 0,5 ul de dNTPs (10 mM). </li><li> Misturar por pipetagem e aquecer a 65 ° C durante 5 min. Em seguida, resfriar lentamente no banco top. </li><li> Depois da reacção ter arrefecido até à temperatura ambiente, coloque o tubo em gelo e adicionar 0,5 ul de cada enzima transcriptase inversa e um inibidor de RNase. Misturar por pipetagem, e colocados no termociclador para o passo de extensão indicado nas instruções do fabricante. </li><li> Uma vez completa suplemento de 5 ul livre de nuclease H2O para levar o volume final da biblioteca de cDNA até 15 ul. </li></ol> 5. Preparação Biblioteca <ol><li> Para a indexação de PCR, montar a seguinte reacção em gelo: 6 ul de 5x Tampão Phusion, 1 ul de dNTPs (10 mM), 0,9 ul de DMSO, 0,75 ul 5 &#39;iniciador (25? M), 0,75 ul iniciador 3&#39; (25? M), biblioteca de cDNA de 3 ul (concentração variável, dependendo da quantidade de entrada), 0,5 ul Phusion polimerase (1 unidade), isento de nuclease de H 2 O para um volume final de 30 ul. Escolha as seqüências de primers para garantir a compatibilidade com o modelo ea plataforma de sequenciamento deescolha. <ol><li> Executar a reacção de PCR com as seguintes definições: desnaturação inicial a 98 ° C durante 3 minutos, durante os primeiros 4 ciclos, desnaturar a 98 ° C durante 15 seg, recozimento a 50 ° C durante 15 seg, estender a 72 ° C durante 15 seg . NOTA: Dependendo da quantidade de material de entrada, variar o total de números de ciclos de PCR. Tipicamente, para uma entrada de 1-2 ug de RNA total, 13 ciclos de PCR no total são suficientes para gerar a biblioteca de miR-Seq. </li><li> Realizar os ciclos subsequentes, por exemplo, ciclos de 5-12, de um modo em duas etapas, onde os ciclos de recozimento e de extensão são combinados e realizados a 70 ° C durante 30 seg e desnaturação é novamente 98 ° C durante 15 seg. NOTA: O volume de reacção é de 30 uL para permitir a pausa do programa de PCR e remover uma alíquota de 10 ul em números de ciclos pré-determinados para monitorizar a aparência do produto desejado. Tipicamente, os ciclos de 10, 13, e 16 são escolhidos para o teste a partir de uma amostra de ARN total. </li></ol></li><li> Preparara 8% não desnaturante em gel de acrilamida por mistura de 2 ml de 40% de acrilamida, 1 ml de 10x TBE, e H 2 O a 10 ml. Em seguida, adicione 100 ml de 10% de APS e 10 ml de TEMED. Despeje gel e deixe set. <ol><li> Pré-correr o gel a 12 V / cm de largura de gel durante 30 min. Poços flush. Adicionar 1 ml de tampão de carregamento de 10x (15% de Ficoll-400, 66 mM EDTA, 20 mM Tris-HCl pH 8,0, 0,1% (w / v) de SDS, 0,01% (w / v) de azul de bromofenol), adicionar 10 ul de amostra a cada poço de dimensões 5 milímetros x 1 mm x 15 milímetros. Normalmente, cada poço pode carregar até 30 l de amostras. Executar o gel de amostra a 10 V / cm de largura de gel em tampão 1 x TBE até que o corante azul de bromofenol apenas corre para fora do fundo do gel. </li><li> Mancha e visualizar gel como descrito no passo 2.3. </li><li> Excisar o par de banda de base 146 e elui-se durante a noite em 0,25 mL de HSCB de uma forma similar ao que é descrito na secção 2.4. Precipitar o DNA num volume igual de isopropanol e 1/10 de volume de acetato de sódio. Lavar o granulado em 70% eThanol e ressuspender em tampão TE. </li><li> Quantificar a biblioteca usando PicoGreen de acordo com as recomendações do fabricante. </li></ol></li></ol>

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The authors declare no competing financial interests.

A metodologia aqui descrita faz uso de diversas variáveis-chave para maximizar a eficiência de ligação, ou seja, altas concentrações de PEG, o uso de ligantes randomizados, e alta concentração de ligantes 2,6,9. Esta abordagem permite que bibliotecas de sequenciamento de forma confiável quantitativos a partir de amostras de RNA total de 2. Nós conduzimos várias titulações de ARN de entrada e concluíram que a metodologia anterior é o mais adequado para quantidades de ARN total na gama...

Metodologias baseadas em alta capacidade de sequenciamento têm sido amplamente aplicada em muitas amostras biológicas nos últimos anos em expandir muito a nossa compreensão da complexidade molecular de sistemas biológicos 3,4. No entanto, a preparação de amostras de RNA de high-throughput seqüenciamento muitas vezes transmite preconceitos específicos inerentes à metodologia empregada, o que limita a utilidade potencial destas técnicas poderosas. Estes método específico polarizações ter sido bem documentado para preparações de biblioteca, de pequena ligadura de ARN baseado em 1,2,5,6. Estes preconceitos resultar em variação mil vezes na lê números de miRNAs sintéticos equimolares, fazendo inferência de abundância miRNA a partir de dados de sequenciamento descontroladamente variável e sujeito a erros. Os estudos focam as propriedades de ligases T4 RNA derivadas de fagos têm documentado que as enzimas exibem preferências à base de nucleótidos 7, que se manifestam como bibliotecas enviesados ​​em experiências de sequenciação de alto rendimento <saté&gt; 1,2,8. A fim de minimizar os preconceitos transmitidos por ligases RNA, várias estratégias têm sido empregadas; macromolecular aglomerando 9, randomizando a sequência de nucleótidos do adaptador que é proximal em relação ao local de ligação 6, e empregando concentrações elevadas de adaptador de ligação 2. Através de uma combinação destes três abordagens temos desenvolvido um fluxo de trabalho para a preparação isenta de bibliotecas de ARN pequenas compatíveis para a sequenciação de alto rendimento (Figura 1). Para comparações diretas entre os protocolos atuais e nosso método otimizado, por favor consulte o nosso recente relatório 2. Este método produz optimizado eficiências de ligação de mais de 95% em ambas as 3 &#39;e 5&#39; etapas e permite a ligação isenta de moléculas de ARN a partir de pequenas amostras biológicas sintéticas e 2.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">3&#39; Linker (5&#39; phosphorylated, 3&#39; blocked)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">5&#39; Linker</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>5&#39; blocked, HPLC Purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL2 (1-249 K227Q)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0351S</td>
			<td>Specialized for ligation of pre-adenylated DNA adapters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (without ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (with ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">RNaseOUT&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Polyethylene Glycol (mol. Wt. 8000)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Nuclease-free water</td>
			<td style="width:175px;">Ambion</td>
			<td style="width:157px;">AM9937</td>
			<td>We have found water collected from a distillation apparatus to be of equvalent quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL1</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Superscript III RT kit</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">18080-051&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Phusion PCR kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0530S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RP1 Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RT Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina Index Primer(s)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">40% Acrylamide</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">BP14081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Urea</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">U6504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ammonium persulfate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tetramethyethylenediamine (TEMED)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">2X Denaturing RNA loading buffer</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Razor blades</td>
			<td style="width:175px;">VWR</td>
			<td style="width:157px;">55411-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">SpinX Centricon Tubes</td>
			<td style="width:175px;">Costar</td>
			<td style="width:157px;">CLS8161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Low Retention Microfuge tubes</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">02-681-320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sybr Gold</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">S-11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Adenylation Kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">E2610L</td>
			<td></td>
		</tr>
	</tbody>
</table>

highly efficient ligation of small rna molecules for microrna quantitation by high-throughput sequencing

Mirna clonagem e sequenciamento de alto rendimento, denominada miR-Seq, assume-se como uma abordagem ampla transcriptoma quantificar miRNAs com resolução de nucleotídeo único. Esta técnica de captura miARNs, anexando os adaptadores 3 &#39;e 5&#39; de oligonucleótidos para moléculas de miARN e permite a descoberta de novo miARN. Acoplamento com plataformas de sequenciamento de próxima geração poderosos, miR-Seq tem sido fundamental para o estudo da biologia miRNA. Contudo, desvios significativos introduzidas por passos de ligação de oligonucleótidos têm impedido o miR-SEQ de ser empregado como uma ferramenta de quantificação precisa. Os estudos anteriores demonstram que polarizações em métodos de miR-Seq actuais conduzem frequentemente a quantificação imprecisa miARN com erros de até 1.000 vezes superior para algumas miARNs 1,2. Para resolver estes preconceitos, transmitidas por meio de ligação de ARN, temos desenvolvido um método de ligação de ARN pequenas que resulta em eficiências de ligação de mais de 95% tanto para 3 &#39;e 5&#39; etapas de ligação. O benchmarking este improvou método de construção da biblioteca utilizando miARNs equimolares sintéticos ou mistos diferencialmente, produz consistentemente lê números com desvio inferior a duas vezes a partir do valor esperado. Além disso, essa alta eficiência método miR-Seq permite preciso de todo o genoma miRNA perfil de in vivo total de amostras de RNA 2.

Os resultados esperados para o método anterior deve, inicialmente, ser observação de uma única troca de nucleótidos (aumento) no tamanho do oligonucleótido de ADN que foi sujeita a adenilação por Mth RNA ligase (Figura 2). Após 3 &#39;ligadura, a visualização do gel de acrilamida indica (ver Figura 3) bandas de peso molecular elevado afiadas evidentes na região de nucleótidos 100-300 do gel. Isto indica que a amostra de ARN total a ser utilizado é de alta qualidad...

Watch this Scientific Journal Video about Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing at JoVE.com

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

Ligadura altamente eficiente de pequeno RNA moléculas microRNA para quantificação por sequenciamento de alto rendimento

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

highly-efficient-ligation-small-rna-molecules-for-microrna

Research

JoVE Journal

Biology

11.8K visualizações.  University of Colorado, Boulder. MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs. 

Vídeo: Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Gramicidina baseado em Ensaio de fluorescência; para determinar Moléculas Pequenas Potencial para Modificação de Propriedades bicamada lipídica

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Biologia

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

High-throughput screening para pequenas moléculas Moduladores de canais de potássio Interiores retificador

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

Geração de substrato para Endonucleases de CRISPR / Cas Sistemas

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA Secundária Previsão Estrutura Usando SHAPE de alta capacidade

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Geração de alta Chromatin Template DNA Immunoprecipitation Qualidade para seqüenciamento de alto rendimento (CHIP-seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Um manual de tela pequena molécula Aproximando de alta capacidade Usando embriões Zebrafish

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Automated Modular High Throughput exopolissacï¿½ido Plataforma de Triagem Juntamente com Análise Carboidratos Fingerprint Highly Sensitive

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-seq: Genoma-wide Mapeamento das Interações Proteína-DNA por High Throughput Sequencing de fragmentos de DNA metilado-Adenina

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Um Protocolo de Avaliação Funcional da Whole-Protein saturação mutagênese Bibliotecas Utilizando-High Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...