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: Es fundamental para mantener las condiciones-RNasa libre durante todo el procedimiento. 1. adenilación de 3 &#39;vinculador <ol><li> Diluir oligonucleótido de ADN diseñada para las reacciones de 3 &#39;de ligación a 100 micras de nucleasa libre de H 2 O. NOTA: El oligonucleótido debe tener un &#39;grupo fosfato, un dinucleótido aleatorizado en el 5&#39; 5 final, y un &#39;didesoxicitosina 3. El 5 &#39;grupo fosfato es un requisito de la enzima para una eficiente Mes 5&#39; adenylation 10. El fosfato puede ser añadido por el pre-tratamiento del oligonucleótido con un polinucleótido quinasa, o pedir con la modificación directamente. El dinucleótido aleatorizado en el extremo 5 &#39;es importante para minimizar la preferencia que ligasas de ARN de exposiciones con certeza de extremo a extremo de unirse a las reacciones sobre otros 6 secuencia. El 3 &#39;didesoxicitosina coloca un elemento de bloqueo en el extremo 3&#39; del oligonucleótido to inhibir la formación de concatámeros enlazador-enlazador durante los pasos posteriores de ligación y también sirve para bloquear el extremo de la molécula de los genes miARN-Enlazador formado en el extremo de 3 &#39;de la reacción de ligación 3 11. La secuencia de engarce oligonucleótido elegido para el método de miR-Seq imparcial es como sigue: 5&#39;PO 4 NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3 &#39;. </li><li> Montar la siguiente reacción adenylation: 200 pmol oligonucleótido 3 &#39;enlazador, 4 l de 10x 5&#39; tampón adenylation ADN, ATP 4 mM l 1, 4 l Mes ARN ligasa, libre de nucleasa H 2 O hasta un volumen final de 40 l. </li><li> Incubar la reacción a 65 ° C durante 1 hora. Terminar reacción por calentamiento a 85 ° C durante 5 min. </li><li> Precipitar enlazador adenylated por la adición de 2,5 volúmenes de etanol al 100% y 1/3 volumen de acetato de amonio 10 M para eliminar ATP residual y para evitar ligaduras no anticipados en futuras reacciones. <ol><li> En pocas palabras, mezclar la colcohol, la sal, y la solución de oligo hasta la homogeneidad, haciendo girar a toda velocidad en una microcentrífuga (~ 15.000 xg) a 4 ° C durante 20 min. Lavar el precipitado en etanol al 70%, secado al aire, y se resuspende en el volumen apropiado de libre de nucleasa H 2 O para producir 50-100 M de oligo adenilado. </li></ol></li><li> Confirmar adenylation mediante la ejecución de un gel de poliacrilamida al 18%. <ol><li> Preparar el gel mezclando juntos la siguiente: 6,3 g de urea, 6,75 ml de 40% de acrilamida, y 1,5 ml de 10x TBE. Añadir H2O y 15 ml. </li><li> Mezclar hasta que la urea se disuelve completamente y añadir 150 l 10% (w / v) de persulfato de amonio (APS) y 15 l tetramethlyethylenediamine (TEMED). </li><li> Una vez que se ha fijado gel, pre-carrera durante 30 min a 24 V / cm de anchura de gel con TBE 1x tampón a temperatura ambiente. Una vez que 30 minutos es hasta los pozos inmediatamente con tampón. </li><li> Mezclar 5 pmol de oligonucleótidos con adenilados y un volumen igual de 2x tampón de desnaturalización del ARN de carga (95% (v / v) formamida,0,02% (w / v) SDS, 0,02% de azul de bromofenol (w / v), 0,01% (w / v) de xileno cianol, EDTA 1,0 mM), calentar brevemente a 75 ° C, y SNAP enfriado en hielo. Prescindir de toda la muestra en el pocillo del gel. También incluya una cantidad idéntica de control no adenilado, y los bajos estándares de tamaño de peso molecular. </li><li> Correr el gel a 24 V / cm hasta que el azul de bromofenol es ¾ de la forma de la parte inferior del gel. Desmontar el aparato, gel post-tinción con solución SYBR-oro en 1x 1x TBE y visualizar el cuadro de UV-transiluminador (Figura 2). NOTA: Gel purificar el oligo adenylated de una manera similar a la descrita anteriormente si &quot;no ARN de entrada&quot; controles están resultando en una alta cantidad de producto de PCR en comparación con las muestras con ARN de entrada. </li></ol></li></ol> 2. vinculador Ligadura a 3 &#39;Fin de miARN <ol><li> 3 &#39;La ligadura <ol><li> Ensamble los componentes siguientes en el hielo en el orden indicado: 1.0 l 50% (w / v) PEG 8000, 0,5 y# 956; l de 10x tampón de ligasa de ARN (500 mM Tris-HCl pH 7,5, 100 mM de MgCl 2, DTT 10 mM), 1,0 l adenylated 3 &#39;Enlazador (100 mM), inhibidor de ARNasa 0,5 l, 0,5-8,0 g RNA total, 0,5 l de ARN ligasa de T4 2, libre de nucleasa H 2 O hasta un volumen final de 5 l. </li><li> Mezclar la reacción mediante pipeteo como la solución es demasiado viscoso para vórtice debido a la alta concentración de PEG 8000. Una vez bien mezclados, coloque la reacción en un termociclador con una tapa calentada. Ajuste el bloque a 16 ° C, y se incuba durante 4 horas. NOTA: La reacción puede hacerse a escala hasta un volumen final de 20 l, sin ninguna pérdida en la eficiencia de la ligadura. Reaccionar durante 4 horas a temperatura ambiente o durante la noche a 4 ° C para producir la eficiencia de ligación esencialmente idénticas. </li></ol></li><li> Purificación en gel <ol><li> Después de 3 &#39;de la ligación, la mezcla de la reacción con un volumen igual de tampón de carga 2x ARN. Calentar a 70 ° C durante 5 min, y broche de presión enfriar en hielo. </li&gt; <li> Ejecutar la muestra en un gel de poliacrilamida al 15% para la purificación PAGE. <ol><li> Preparar un gel de poliacrilamida al 15% que contiene urea 7 M, en una manera similar a la descrita previamente en el paso 1.5. </li><li> Pre-correr el gel a 24 V / cm de anchura de gel por lo menos durante 30 minutos. Apague la fuente de alimentación, los pozos de descarga, carga y muestra de gel de carrera en mismo voltaje que antes de la carrera. </li><li> Cargue la muestra y ejecutar la página hasta que el azul de bromofenol sólo ha salido del gel. El producto deseado migrará cerca de la cianol xileno. </li></ol></li></ol></li><li> La tinción, visualización, y Escisión <ol><li> Desmontar aparato de gel y el lugar de gel en la bandeja limpia con 1x tampón (TBE), 1x SYBR-oro, y roca durante 15 min. </li><li> Retire gel de placa de tinción y visualizar el cuadro de transiluminador UV cubierta con una envoltura de plástico limpio. El producto de ligación debe ser alrededor de 45 nucleótidos de longitud. </li><li> Impuestos especiales de la región que contiene el producto de ligación con un razo limpiohoja r y colocar en 0,5 ml tubo de micro-centrífuga. </li></ol></li><li> El aislamiento del producto y Precipitación <ol><li> Pierce inferior de 0,5 ml tubo de microcentrífuga con una aguja de 30 G y colocar el tubo perforado en un segundo, baja retención de limpia, de 1,5 ml tubo de microcentrífuga. Centrifugar durante 5 min a ~ 15.000 x g. </li><li> Visualmente confirmar que toda la porción de gel se ha movido a través del agujero en el tubo interior. Si es necesario, utilice una punta de pipeta limpia para empujar algunos fragmentos de gel más cerca del agujero. </li><li> Desechar el tubo 0,5 ml vacío y añadir 400 l de tampón de la columna de alta sal (HSCB NaCl 400 mM, Tris-HCl 25 mM, pH 7,5, 0,1% SDS) a la suspensión de acrilamida. Tubo (s) de la roca a 4 ° C durante la noche. </li><li> Transferencia de lechada a una columna de centrifugación de acetato de celulosa de 0,22 micras utilizando una punta de pipeta de gran calibre. Centrifugar durante 3 min a temperatura ambiente a ~ 15.000 xg para recoger todo el líquido lejos de matriz de gel de acrilamida. </li><li> Trasvasar el líquido a una, bajo la retención limpia, tubo de 1,5 ml contieneing 1 l de 20 mg / ml de glucógeno. Añadir un volumen igual de isopropanol y 1/10 de acetato de sodio volumen XX. Mezclar solución invirtiendo tubo varias veces y precipitar en -80 ° C congelador durante 20 min. </li><li> Centrifugar a toda velocidad a 4 ° C durante 20 min para sedimentar el ácido nucleico. Eliminar el sobrenadante y lavar en etanol al 70%. Aire pellet seco durante 10 min a temperatura ambiente y proceder directamente a 5 &#39;etapa de ligación. </li></ol></li></ol> 3. vinculador ligadura de extremo 5 &#39;de los genes miARN-3&#39; Enlazador híbrido <ol><li> Para ácido nucleico granulado, añadir los siguientes componentes: 2 l de PEG 8000 (50% w / v), 0,5 l de 10x RNA Ligase Buffer, 0,5 l ATP (10 mM), 0,5 l NN-ARN oligo (100 mM), y H 2 O hasta un volumen final de 4 l. NOTA: La composición de la &#39;NN-ARN oligo&#39; es el siguiente 5 &#39;invertida-didesoxi-TCUACAGUCCGACGAUCNN3&#39; y se purificó por medio de HPLC por el fabricante. </li><li> Mezclaresta muestra pipeteando arriba y abajo varias veces. Haga esto durante algún tiempo (hasta 5 min) para asegurarse de que el sedimento ha sido re-solubilizado por completo. Si re-solubilización es problemático, calentar la muestra brevemente (3-5 min) a 37 ° C y reanudar pipeteado para mezclar. </li><li> Una vez que la pastilla está de vuelta en solución, transferir el contenido de un tubo de PCR limpio que contiene 1 l de una mezcla 1: 1 de inhibidor de RNasa y T4 ARN ligasa 1 (5 unidades). Una vez más la mezcla pipeteando arriba y abajo. </li><li> Incubar la reacción en un ciclador térmico a 37 ° C durante 4 h. Proceda inmediatamente a la síntesis de ADNc. </li></ol> 4. Transcripción inversa / cDNA Synthesis <ol><li> Añadir los siguientes componentes a los 5 &#39;reacción de ligación: 2 l de tampón 5x RT, 0,75 l de DTT 100 mM, 1 l Illumina RT Primer 5&#39;-GCCTTGGCACCCGAGAATTCCA-3&#39; (1 M), y 0,5 mu l dNTPs (10 mM). </li><li> Mezclar mediante pipeteo y se calienta a 65 ° C durante 5 min. Luego enfriar lentamente en el banco top. </li><li> Una vez que la reacción se ha enfriado a temperatura ambiente, colocar el tubo en hielo y añadir 0,5 l de cada uno de enzima transcriptasa inversa y un inhibidor de RNasa. Mezclar mediante pipeteo y el lugar en el termociclador para la etapa de extensión se indica en las instrucciones del fabricante. </li><li> Una vez completa add 5 l de nucleasa libre de H 2 O para llevar el volumen final de la biblioteca de ADNc de hasta 15 l. </li></ol> 5. Biblioteca Preparación <ol><li> Para la indexación PCR, montar la siguiente reacción en hielo: 6 l 5x Phusion Buffer, 1 mu l dNTPs (10 mM), 0,9 l de DMSO, 0,75 l Cebador 5 &#39;(25 mM), 0,75 l cebador 3&#39; (25 mM), biblioteca de ADNc de 3 l (concentración variable en función de la cantidad de la entrada), 0,5 l de la polimerasa Phusion (1 unidad), libre de nucleasa H 2 O hasta un volumen final de 30 l. Elegir las secuencias de cebadores para asegurar la compatibilidad con la plantilla y la plataforma de secuenciación deelección. <ol><li> Ejecutar la reacción de PCR con los siguientes ajustes: desnaturalización inicial a 98 ° C durante 3 min, durante los primeros 4 ciclos, desnaturalizar 98 ° C durante 15 s, recocido a 50 ° C durante 15 s, se extienden a 72 ° C durante 15 s . NOTA: En función de la cantidad del material de entrada, varía el número total de ciclos de PCR. Típicamente, para una entrada de 1-2 g de RNA total, 13 ciclos totales de PCR son suficientes para generar la biblioteca de miR-Seq. </li><li> Llevar a cabo los ciclos posteriores, por ejemplo, los ciclos 5-12, de una manera en dos etapas, donde se combinan y se llevaron a cabo a 70 ° C durante 30 segundos y la desnaturalización de los ciclos de recocido y extensión es de nuevo 98 ° C durante 15 s. NOTA: El volumen de reacción es de 30 l para permitir una pausa en el programa de PCR y retirar una alícuota de 10 l en los números de ciclo pre-determinados para monitorizar la apariencia del producto deseado. Típicamente, los ciclos 10, 13, y 16 son elegidos para las pruebas de una muestra de ARN total. </li></ol></li><li> Prepararun no desnaturalización gel de acrilamida al 8% mediante la mezcla de 2 ml de 40% de acrilamida, 1 ml de TBE 10x, y H 2 O a 10 ml. A continuación, añadir 100 l de 10% de APS y 10 l de TEMED. Vierta en gel y dejar establecido. <ol><li> Pre-correr el gel a 12 V / cm de anchura de gel durante 30 min. Lavar los pozos. Añadir 1 l de 10x tampón de carga (15% de Ficoll-400, EDTA 66 mM, 20 mM Tris-HCl pH 8,0, 0,1% (w / v) SDS, 0,01% (w / v) de azul de bromofenol), añadir 10 l de muestra a cada pocillo de dimensiones 5 mm x 1 mm x 15 mm. Por lo general, cada pozo puede cargar hasta 30 l de muestras. Ejecutar la muestra de gel a 10 V / cm de anchura de gel en tampón 1x TBE hasta que el colorante azul de bromofenol sólo se ejecuta fuera de la parte inferior del gel. </li><li> Mancha y visualizar en gel como se describe en el paso 2.3. </li><li> Escindir la banda de par 146 base y eluir durante la noche en 0,25 ml de HSCB de una manera similar a lo que se describe en la sección 2.4. Precipitado de ADN en un volumen igual de isopropanol y acetato de décimo volumen de sodio XX. Lavar la pella en el 70% eTHANOL y resuspender en tampón TE. </li><li> Cuantificar la biblioteca utilizando PicoGreen de acuerdo con las recomendaciones del fabricante. </li></ol></li></ol>

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The authors declare no competing financial interests.

La metodología descrita en este documento hace uso de varias variables clave para maximizar la eficiencia de ligación, a saber, las altas concentraciones de PEG, el uso de enlazadores aleatorios, y alta concentración de enlazadores 2,6,9. Este enfoque permite a las bibliotecas de secuenciación de forma fiable cuantitativos de muestras de ARN total de 2. Hemos llevado a cabo múltiples titulaciones de ARN de entrada y han concluido que la metodología anterior es el más adecuado para las cantid...

Metodologías basadas secuenciación de alto rendimiento han sido ampliamente aplicado a muchas muestras biológicas en los últimos años en expansión en gran medida nuestra comprensión de la complejidad molecular de los sistemas biológicos 3,4. Sin embargo, la preparación de muestras de ARN de secuenciación de alto rendimiento a menudo imparte sesgos específicos inherentes a la metodología empleada, lo que limita la utilidad potencial de estas técnicas de gran alcance. Estos sesgos de métodos específicos han sido bien documentados para las preparaciones de bibliotecas basado en la ligadura, pequeño-ARN 1,2,5,6. Estos sesgos como resultado la variación de 1.000 veces en lee los números de miRNAs sintéticos equimolares, hacer inferencia de miARN abundancia a partir de datos de secuenciación tremendamente variable y propenso a errores. Estudios centrados en las propiedades de T4 ARN ligasas derivadas de fagos han documentado que las enzimas exhiben preferencias basadas en nucleótidos 7, que se manifiestan como bibliotecas sesgadas en experimentos de secuenciación de alto rendimiento <sarriba&gt; 1,2,8. A fin de minimizar los sesgos impartidas por ARN ligasas, múltiples estrategias se han empleado; macromolecular crowding 9, la aleatorización de la secuencia de nucleótidos en el adaptador que es proximal al sitio de la ligadura de 6, y el empleo de altas concentraciones de adaptador de ligadura 2. A través de una combinación de estos tres enfoques, hemos desarrollado un flujo de trabajo para la preparación imparcial de pequeñas bibliotecas de ARN compatibles para la secuenciación de alto rendimiento (Figura 1). Para las comparaciones directas entre los protocolos actuales y nuestro método optimizado, por favor consulte nuestro informe reciente 2. Este método produce optimizado la eficiencia de ligación de más del 95% tanto en 3 &#39;y 5&#39; pasos y permite la ligadura imparcial de pequeñas moléculas de ARN de las muestras sintéticas y biológicas 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 clonación y secuenciación de alto rendimiento, denominado miR-Sec, destaca como un enfoque de todo el transcriptoma de cuantificar miRNAs con resolución de un solo nucleótido. Esta técnica captura miRNAs por fijar los adaptadores 3 &#39;y 5&#39; de oligonucleótidos a las moléculas de miARN y permite de novo miARN descubrimiento. El acoplamiento con potentes plataformas de secuenciación de próxima generación, el miR-Sec ha sido fundamental en el estudio de la biología de miRNA. Sin embargo, los sesgos significativos introducidos por pasos de ligación de oligonucleótidos han impedido que el miR-Sec de ser empleado como una herramienta de cuantificación exacta. Estudios previos demuestran que los sesgos en los métodos de miR-Seq actuales a menudo conducen a la cuantificación inexacta miARN con errores hasta 1.000 veces por algunos miRNAs 1,2. Para resolver estos sesgos impartidas por la ligadura de ARN, hemos desarrollado un pequeño método de ligadura de ARN que se traduce en eficiencias de ligación de más del 95% para ambos 3 &#39;y 5&#39; de ligación pasos. Evaluación comparativa de este improbado método de construcción de la biblioteca utilizando miRNAs sintéticos equimolares o diferencialmente mixtos, produce consistentemente lee los números con una desviación de menos de dos veces del valor esperado. Por otra parte, este método de gran eficacia miR-Sec permite precisa de todo el genoma de perfiles de miARN in vivo total de muestras de ARN 2.

Los resultados esperados por el método anterior deben ser inicialmente la observación de un solo turno de nucleótidos (aumento) en el tamaño del oligonucleótido de ADN que fue objeto de adenylation por Mes ARN ligasa (Figura 2). Después de 3 &#39;de la ligación, la visualización del gel de acrilamida indica (véase la Figura 3) bandas de peso molecular alto afilados evidente en la región de nucleótidos 100-300 de la gel. Esto indica que la muestra de ARN total que se ...

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 los Pequeños ARN moléculas de microARN cuantificación mediante secuenciación de alto rendimiento

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 vistas.  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. 

Video: 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 basado en ensayo de fluorescencia, para la determinación de moléculas pequeñas posibles para modificar las propiedades bicapa lipídica

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

Investigación

Biología

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.

De alto rendimiento para los moduladores de molécula pequeña de tráfico de perfeccionamiento activo Canales rectificador de potasio

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.

Sustrato Generación para endonucleasas 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.

ARN de predicción de estructura secundaria Utilizando SHAPE de alto rendimiento

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.

Generación de Plantilla ADN Immunoprecipitation cromatina alta calidad para la secuenciación de alto rendimiento (chip-ss)

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.

Un manual de la pantalla pequeña molécula Acercarse De Alto Rendimiento Utilizando embriones de pez cebra

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.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

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.

Plataforma modular de selección automatizados de alto rendimiento de exopolisacáridos Junto con el análisis de carbohidratos de la huella digital de alta sensibilidad

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 siguientes: Genoma de toda la cartografía de la proteína-ADN Interacciones de alto rendimiento de secuenciación de fragmentos de ADN adenina-metilado

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.

Un protocolo para la evaluación funcional de la Proteína Total-mutagénesis de saturación Bibliotecas La utilización de secuenciación de alto rendimiento

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