C.Y.W. received support from the National Health Research Institute (NHRI-EX101-9925SC), the National Science Council (101-2314-B-182-100-MY3, 101-2314-B-182A-009), and Chang Gung Memorial Hospital (CMRPG3B1643, CMRPG3D1002, CMRPG3D0581, CMRPG380091, and CMRPG3C1763).

NOTA: El ARN total m 6 A nivel incluye ARNr, ARNm, y otros ARN pequeños. Puesto que el ARN ribosomal tiene abundantes m 6 modificaciones A, la medición de los niveles A 6 m deberán tener en cuenta este hecho. 1. Aislamiento de ARN <ol><li> El uso de la solución de descontaminación ribonucleasa (pulveriza sobre toallas de papel), limpie las pipetas y las superficies de mesa de laboratorio. toallas de papel mojadas con agua libre de nucleasa y limpiar las pipetas y las superficies de mesa de laboratorio de nuevo. </li><li> La extracción de RNA <ol><li> Aislamiento de ARN total <ol><li> El uso de un agitador de cabeza, homogeneizar 50-100 mg de tejido o de 5-10 x 10 6 células en 1 ml de solución de aislamiento de ARN mantenidas a 4 ° C. NOTA: Más de tejido (100-150 mg) pueden ser necesarios para las muestras de tejido adiposo de ratones debido a las concentraciones de ARN más bajos en el mismo. Las muestras proceden de corazón de ratón, hígado, músculo esquelético, pulmón, cerebro, y los macrófagos se han empleado previamente 4. </li><li> EnCubate los homogeneizados de muestra durante 5 min a temperatura ambiente. </li><li> Añadir 100 ml de 1-bromo-3-cloropropano (BCP) por 1 ml de homogeneizado de la muestra. Agitar los tubos vigorosamente durante 15 s y proceder a aislar RNA total de acuerdo con las instrucciones del fabricante 24. </li></ol></li><li> Poliadenilado de purificación de ARNm a partir de ARN total aislado <ol><li> Purificar el ARNm poliadenilado utilizando kits o protocolos disponibles. <ol><li> Agitar a fondo para volver a suspender cada una de las siguientes soluciones: solución de enlace, perlas de poliestireno oligo (dT), y solución de lavado de 2x. Asegúrese de que el oligo (dT) Cuentas de calentar a temperatura ambiente antes de su uso. </li><li> Transferir 120 l de solución de elución por la preparación en un tubo y se calienta a 70 ° C en un bloque de calentamiento. </li><li> Pipetear hasta 500 g de ARN total en un tubo de microcentrífuga. Con agua libre de nucleasa, ajustar el volumen a 250 mL. </li><li> Añadir 250 l de solución de enlace 2x con el ARN total, de modolución y vortex brevemente para mezclar el contenido. </li><li> Añadir 15 l de oligo (dT) perlas y mezclar bien para mezclar. </li><li> Incubar la mezcla a 70 ° C durante 3 min. </li><li> Sacar la muestra del bloque de calentamiento y se deja reposar a temperatura ambiente durante 10 min. </li><li> Se centrifuga a 15.000 xg durante 2 min. </li><li> Retirar con cuidado el sobrenadante, dejando atrás aproximadamente 50 l. </li><li> Añadir 500 l de solución de lavado a una nueva suspensión del sedimento con la pipeta. </li><li> Pipetear la suspensión en un conjunto tubo de filtro giratorio / colección. </li><li> Se centrifuga a 15.000 xg durante 2 min. Retirar la columna del tubo de recogida y desechar el flujo a través. Retorno de la columna para el tubo de recogida. </li><li> Pipetear 500 l de solución de lavado sobre el filtro de centrifugado. </li><li> Centrifugar a 15.000 x g durante 2 min. </li><li> Transferir el filtro giratorio en un tubo de recogida fresca. </li><li> Pipetear 50 l de solución de elución calientaa 70 ° C en el centro del filtro de centrifugado. </li><li> Incubar durante 2-5 minutos a 70 ° C. Se centrifuga a 15.000 xg durante 1 min. </li><li> Pipetear un 50 l adicionales de solución de elución se calienta a 70 ° C en el centro del filtro de centrifugado. </li><li> Repita el paso 1.2.2.1.18. </li></ol></li><li> Añadir 100 g / ml de glucógeno, 0,1 volumen de tampón de sodio 3 M de acetato (pH 5,2) y 2,5 volúmenes de etanol absoluto. Precipitar durante la noche a -80 ° C. </li><li> Centrifugar a 15.000 xg durante 25 min a 4 ° C. Descartar el sobrenadante. </li><li> Lavar el sedimento con 1 ml de 75% de etanol. Centrifugar a 15.000 xg durante 15 min a 4 ° C. Retirar con cuidado el etanol. </li><li> Secar al aire durante 3-5 min. </li><li> Añadir 10 l de agua libre de nucleasa a disolver el precipitado. </li><li> Almacenar a -70 ° C. </li></ol></li></ol></li><li> Calificación y Cuantificación de ARN <ol><li> El uso de un espectrofotómetro, determinar la concentración de ARN por NotIng de la absorbancia a 260 nm y 280 nm. La relación A 260 / A 280 debería ser 1/8 a 2/2. </li><li> Comprobar la calidad de las muestras de ARN mediante electroforesis en gel de agarosa al 0,8% 25. NOTA: El ARN total eucariota intacta debería mostrar intensas bandas 28S y 18S rRNA. La relación de 28S frente a la intensidad de banda 18S rRNA para una buena preparación de ARN es ~ 2. </li></ol></li></ol> 2. Electroforesis en gel y transferencia NOTA: Los protocolos de preparación de tampón se dan en la Tabla 1. <ol><li> El formaldehído Preparación del gel (1%) <ol><li> Enjuague todo el equipo de electroforesis con agua diethylpyrocarbonate (DEPC). </li><li> Derretir 2,5 g de agarosa en 215 ml de agua DEPC completamente en un horno de microondas. </li><li> Añadir 12,5 ml de 10x 3- (N- morfolino) propanosulfónico (MOPS) tampón y 22,5 ml de formaldehído al 37%. </li><li> Se vierte en el aparato de electroforesis con un peine de espesor 1,5 mm. </li><li> Eliminar las burbujas o empujar tdobladillo a los bordes del gel con un peine limpia. </li><li> Permitir que el gel solidifique a temperatura ambiente. </li></ol></li><li> Preparación de la muestra <ol><li> Mezclar 1-10 g de ARN total y 11,3 l de tampón de muestra. </li><li> Mezclar 2 g del marcador de ARN (1 g / l) con 11,3 l de tampón de la muestra. </li><li> Añadir agua libre de nucleasa a las muestras de 2.2.1 y 2.2.2 a un volumen total de 16 mL. </li><li> Se calienta a 60 ° C durante 5 min, y luego se enfría en hielo. </li><li> Mezclar 16 l de la muestra a partir de 2.2.3 con 4 l de colorante de seguimiento, que contiene 0,1 g / l de bromuro de etidio en hielo. PRECAUCIÓN: El bromuro de etidio es teratogénico y posiblemente tóxicos si se inhalan. Leer bromuro de etidio hoja técnica de seguridad. </li></ol></li><li> electroforesis <ol><li> Añadir 200 ml de 10x MOPS buffer de 1.800 ml de ddH2O para hacer un tampón de desarrollo 1x MOPS. </li><li> Utilice el tampón de MOPS 1x para enjuagar los pocillos del gel. </li><li> Previa al funcionamiento de tél gel a 20 V durante 5 min. </li><li> Cargar las muestras de ARN en los pocillos del gel. </li><li> Cubrir con papel de aluminio para evitar la exposición a la luz. Ejecutar a 35 V durante aproximadamente 17 h (durante la noche) </li><li> Examinar y fotografiar el gel bajo una luz ultravioleta. </li></ol></li><li> Transferir <ol><li> Cortar el gel para eliminar la parte no utilizada. </li><li> Preparar 500 ml de tampón 10x SSC (250 ml de 20x tampón SSC y 250 ml de agua DEPC). </li><li> Se lava el gel dos veces con 10x tampón SSC durante 20 min con agitación a 50 rpm. </li><li> Cortar la hoja de papel 1 de filtro suficientemente grande para servir como un documento de mecha, que absorbe tampón de transferencia de la bandeja de transferencia (Figura 1). </li><li> Cortar 4 trozos de papel de filtro para el mismo tamaño que el gel. </li><li> Cortar 1 hoja de membrana de nylon cargada positivamente con el mismo tamaño que el gel. </li><li> Marque una muesca en el gel y la membrana como un marcador para asegurar la orientación apropiada. </li><li> Remojar la membrana en tampón SSC 2x durante 15 minutos. </li><li> Verter 500 ml de Of 10x SSC tampón en la bandeja de transferencia. </li><li> Ponga la hoja de papel de filtro grande a través de la placa de vidrio y humedecer el papel de filtro con tampón de transferencia. </li><li> Estirar las burbujas con una pipeta. </li><li> Remojar 2 piezas de papel de filtro de pre-corte en tampón de transferencia y colocarlos en el centro del papel de filtro de paso 2.4.5. Eliminar las burbujas. </li><li> Coloque el lado superior del gel en el papel de filtro. </li><li> Colocar la membrana en la parte superior del gel. Alinear las muescas del gel y la membrana. </li><li> Colocar 2 piezas de papel de filtro de pre-corte en la parte superior de la membrana y humedecer el papel de filtro con tampón de transferencia. </li><li> Eliminar las burbujas entre las capas. </li><li> Aplicar una envoltura de plástico alrededor del gel para asegurar que las toallas solamente absorben tampón que pasa por el gel y la membrana. </li><li> Pila de las toallas de papel en la parte superior del papel de filtro. </li><li> Colocar una placa de vidrio de gran tamaño en la parte superior de las toallas. </li><li> Coloque un peso en la parte superior de la pila. </li><li>Mantener durante la noche para permitir que el ARN para transferir desde el gel a la membrana. </li></ol></li></ol> 3. Detección de N 6 -methyladenosine <ol><li> La reticulación de la membrana <ol><li> Mantener la membrana húmeda en tampón SSC 2x. </li><li> Coloque 2 hojas de papel de filtro sumergido con tampón 10x SSC en agente de reticulación UV. </li><li> Colocar la membrana en la parte superior del papel de filtro, con el lado de ARN adsorbido hacia arriba. </li><li> Seleccionar &quot;modo autocrosslink&quot; (120.000 mu J) y pulse el botón &quot;Inicio&quot; para iniciar la irradiación. </li><li> Examinar la membrana y el gel con una imagen gel receptor UV para confirmar la transferencia del ARN del gel a la membrana. </li></ol></li><li> inmunotransferencia <ol><li> Bloquear la membrana en la leche no grasa 5% en solución salina tamponada con Tris con 20 tampón de Tween (TBST) a temperatura ambiente durante 1 hr. </li><li> Lavar tres veces con tampón de TBST durante 15 min. </li><li> Incubar la membrana durante la noche en m 6 A (&lt;em&gt; solución de N 6 -methyladenosine) de anticuerpos (1: no grasa de la leche TBST buffer 1.000 en 5%) a 4 ° C. </li><li> Se lava la membrana tres veces con tampón de TBST durante 15 min. </li><li> Incubar la membrana en la solución de burro conjugado con HRP anti-anticuerpo de conejo (1: 2000 en tampón TBST leche 5% no grasa) durante 1 h a temperatura ambiente. </li><li> Se lava la membrana tres veces con tampón de TBST durante 15 min. </li><li> Aplicar el sustrato quimioluminiscente intensificado (0,125 ml por cm2 de membrana). </li><li> Capturar la quimioluminiscencia con los ajustes óptimos de la cámara digital, de acuerdo con las instrucciones del fabricante 26. </li></ol></li><li> m 6 una cuantificación <ol><li> Medir la m 6 Una intensidad de quimioluminiscencia relativa utilizando el software ImageJ. <ol><li> Bajo el menú del software ImageJ, seleccione la opción &quot;Archivo&quot; para abrir el archivo pertinente. </li><li> Seleccione la herramienta &quot;Rectángulo&quot; de ImageJ y dibujar un marco alrededorlas señales. </li><li> Si RNA ribosomal no es el foco de los experimentos, evitar los 18S y 28S ARN ribosomal m 6 bandas A para el cálculo. </li><li> Haga clic en &quot;Comando&quot; y &quot;1&quot; para confirmar el carril seleccionado. </li><li> Pulse la tecla &quot;Comando&quot; y &quot;3&quot; para mostrar la parcela seleccionada. </li><li> Haga clic en la herramienta &quot;Straight&quot; y trazar líneas para separar el área segmentada. </li><li> Haga clic en la herramienta &quot;varita&quot; para registrar las mediciones. </li><li> Exportar los datos. </li><li> Normalizar los niveles de m 6 A con el 18S ARN ribosomal banda de la tinción con bromuro de etidio. </li></ol></li></ol></li></ol>

<ol>
	<li>Thammana, P., Held, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation+of+16S+RNA+during+ribosome+assembly+in+vitro.">Methylation of 16S RNA during ribosome assembly in vitro.</a> Nature. 251 (5477), 682-686 (1974).</li><li>Meyer, K. D., Jaffrey, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+epitranscriptome:+N6-methyladenosine+and+gene+expression+control.">The dynamic epitranscriptome: N6-methyladenosine and gene expression control.</a> Nat Rev Mol Cell Biol. 15 (5), 313-326 (2014).</li><li>Niu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N6-methyl-adenosine+(m6A)+in+RNA:+an+old+modification+with+a+novel+epigenetic+function.">N6-methyl-adenosine (m6A) in RNA: an old modification with a novel epigenetic function.</a> Genomics Proteomics Bioinformatics. 11 (1), 8-17 (2013).</li><li>Wang, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+FTO+in+adipose+tissue+decreases+Angptl4+translation+and+alters+triglyceride+metabolism.">Loss of FTO in adipose tissue decreases Angptl4 translation and alters triglyceride metabolism.</a> Sci Signal. 8 (407), 127 (2015).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N6-methyladenosine-dependent+regulation+of+messenger+RNA+stability.">N6-methyladenosine-dependent regulation of messenger RNA stability.</a> Nature. 505 (7481), 117-120 (2014).</li><li>Berulava, T., Rahmann, S., Rademacher, K., Klein-Hitpass, L., Horsthemke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N6-adenosine+methylation+in+MiRNAs.">N6-adenosine methylation in MiRNAs.</a> PLoS One. 10 (2), 0118438 (2015).</li><li>Deng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+occurrence+of+N6-methyladenosine+in+bacterial+mRNA.">Widespread occurrence of N6-methyladenosine in bacterial mRNA.</a> Nucleic Acids Res. 43 (13), 6557-6567 (2015).</li><li>Dominissini, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+N-methyladenosine+methylome+in+eukaryotic+messenger+RNA.">The dynamic N-methyladenosine methylome in eukaryotic messenger RNA.</a> Nature. , (2016).</li><li>Fustin, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-methylation-dependent+RNA+processing+controls+the+speed+of+the+circadian+clock.">RNA-methylation-dependent RNA processing controls the speed of the circadian clock.</a> Cell. 155 (4), 793-806 (2013).</li><li>Geula, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells.+m6A+mRNA+methylation+facilitates+resolution+of+naive+pluripotency+toward+differentiation.">Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.</a> Science. 347 (6225), 1002-1006 (2015).</li><li>Wang, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FTO+modulates+fibrogenic+responses+in+obstructive+nephropathy.">FTO modulates fibrogenic responses in obstructive nephropathy.</a> Sci Rep. , (2016).</li><li>Jia, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N6-methyladenosine+in+nuclear+RNA+is+a+major+substrate+of+the+obesity-associated+FTO.">N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.</a> Nat Chem Biol. 7 (12), 885-887 (2011).</li><li>Du, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+association+study+of+the+m6A+genes+with+major+depressive+disorder+in+Chinese+Han+population.">An association study of the m6A genes with major depressive disorder in Chinese Han population.</a> J Affect Disord. 183, 279-286 (2015).</li><li>Wang, C. Y., Yeh, J. K., Shie, S. S., Hsieh, I. C., Wen, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+RNA+N6-methyladenosine+and+the+role+of+cryptochrome.">Circadian rhythm of RNA N6-methyladenosine and the role of cryptochrome.</a> Biochem Biophys Res Commun. 465 (1), 88-94 (2015).</li><li>Wang, C. Y., Shie, S. S., Hsieh, I. C., Tsai, M. L., Wen, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FTO+modulates+circadian+rhythms+and+inhibits+the+CLOCK-BMAL1-induced+transcription.">FTO modulates circadian rhythms and inhibits the CLOCK-BMAL1-induced transcription.</a> Biochem Biophys Res Commun. 464 (3), 826-832 (2015).</li><li>Heyn, H., Esteller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Adenine+Code+for+DNA:+A+Second+Life+for+N6-Methyladenine.">An Adenine Code for DNA: A Second Life for N6-Methyladenine.</a> Cell. 161 (4), 710-713 (2015).</li><li>Xiao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+m(6)A+Reader+YTHDC1+Regulates+mRNA+Splicing.">Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.</a> Mol Cell. 61 (4), 507-519 (2016).</li><li>Meyer, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+analysis+of+mRNA+methylation+reveals+enrichment+in+3'+UTRs+and+near+stop+codons.">Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.</a> Cell. 149 (7), 1635-1646 (2012).</li><li>Horowitz, S., Horowitz, A., Nilsen, T. W., Munns, T. W., Rottman, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+N6-methyladenosine+residues+in+bovine+prolactin+mRNA.">Mapping of N6-methyladenosine residues in bovine prolactin mRNA.</a> Proc Natl Acad Sci U S A. 81 (18), 5667-5671 (1984).</li><li>Resnick, R. J., Noreen, D., Munns, T. W., Perdue, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+N6-methyladenosine+in+expression+of+Rous+sarcoma+virus+RNA:+analyses+utilizing+immunoglobulin+specific+for+N6-methyladenosine.">Role of N6-methyladenosine in expression of Rous sarcoma virus RNA: analyses utilizing immunoglobulin specific for N6-methyladenosine.</a> Prog Nucleic Acid Res Mol Biol. 29, 214-218 (1983).</li><li>Golovina, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+site-specific+detection+of+m6A+nucleoside+presence+in+RNA+based+on+high-resolution+melting+(HRM)+analysis.">Method for site-specific detection of m6A nucleoside presence in RNA based on high-resolution melting (HRM) analysis.</a> Nucleic Acids Res. 42 (4), 27 (2014).</li><li>Dominissini, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topology+of+the+human+and+mouse+m6A+RNA+methylomes+revealed+by+m6A-seq.">Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.</a> Nature. 485 (7397), 201-206 (2012).</li><li>Linder, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-nucleotide-resolution+mapping+of+m6A+and+m6Am+throughout+the+transcriptome.">Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.</a> Nat Methods. 12 (8), 767-772 (2015).</li><li>Fu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Quantification+of+Methylated+Cytidine+and+Adenosine+in+Cellular+and+Tissue+RNA+by+Nano-Flow+Liquid+Chromatography-Tandem+Mass+Spectrometry+Coupled+with+the+Stable+Isotope-Dilution+Method.">Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.</a> Anal Chem. 87 (15), 7653-7659 (2015).</li><li>Rio, D. C., Ares, M., Hannon, G. J., Nilsen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondenaturing+agarose+gel+electrophoresis+of+RNA.">Nondenaturing agarose gel electrophoresis of RNA.</a> Cold Spring Harb Protoc. 2010 (6), 5445 (2010).</li><li>Wang, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FTO+modulates+fibrogenic+responses+in+obstructive+nephropathy.">FTO modulates fibrogenic responses in obstructive nephropathy.</a> Sci Rep. 6, 18874 (2016).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+mapping+reveals+reversible+and+dynamic+N-methyladenosine+methylome.">Transcriptome-wide mapping reveals reversible and dynamic N-methyladenosine methylome.</a> Nat Chem Biol. , (2016).</li><li>Sanschagrin, S., Yergeau, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing+of+16S+ribosomal+RNA+gene+amplicons.">Next-generation sequencing of 16S ribosomal RNA gene amplicons.</a> J Vis Exp. (90), (2014).</li><li>Kukutla, P., Steritz, M., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+ribosomal+RNA+for+mosquito+gut+metagenomic+RNA-seq.">Depletion of ribosomal RNA for mosquito gut metagenomic RNA-seq.</a> J Vis Exp. (74), (2013).</li><li>Mishima, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immuno-Northern+Blotting:+Detection+of+RNA+Modifications+by+Using+Antibodies+against+Modified+Nucleosides.">Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.</a> PLoS One. 10 (11), 0143756 (2015).</li><li>Klungland, A., Dahl, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+RNA+modifications+in+disease.">Dynamic RNA modifications in disease.</a> Curr Opin Genet Dev. 26, 47-52 (2014).</li></ol>

Los autores no tienen nada que revelar.

Modifications of RNA have important roles in cellular function and physiology. The current understanding of the regulation, function, and homeostasis of these modifications is still being explored and expanded8. Therefore, a precise and gold-standard method to evaluate the modifications of RNA is needed. The modified northern blotting method provides precise quantification of RNA modifications and clear delineation of the modifications in diverse RNAs. Although the method requires at least 3 days, it can be standardized and can be used in various experimental designs. Moreover, with different antibodies, it can detect different RNA modifications27.
It is important to separate different RNAs when analyzing RNA modifications. Ribosomal RNA comprises a large portion of the total amount of RNA28,29. The results from analyzing RNA modifications only in total RNA will represent mostly the changes of rRNA. Methylation and other such modifications of rRNA could potentially mask the changes in other RNAs. With the procedure of gel separation, the modifications of mRNA and other small RNAs can be more accurately analyzed.
Transcriptome-wide mapping with m6A immunoprecipitation and sequencing provides detailed insight into the modification of each type of RNA22. It provides information on the specific RNAs and a resolution of around 80-120 bp. Although m6A-Seq can compare the modifications between different experimental conditions, the selection of proper standards and controls for such experiments is difficult18. Immunoprecipitation is difficult to reproduce, often giving significant variations amongst repeats. Moreover, m6A-Seq requires the fragmentation of RNA samples before immunoprecipitation and sequencing30. The fragmentation process could potentially induce undue influences on the original RNA modifications. If the experiment does not need the specific gene's information but requires different conditions for comparison, the current method provides better visualization and control for diverse experimental setups. 
Modification of RNA is an important step in regulating transcriptional control31. However, the homeostasis and the regulatory mechanisms of various RNA modifications under diverse physiological realms are still unclear. Using the present modified northern blotting method, different RNA modifications can be quantified and compared. Furthermore, the changes and regulations of RNA modifications can be investigated in greater detail. In the future, it could also be possible to combine the experimental data from both the classical northern blotting and the modified northern blotting protocols, providing greater insights into RNA biology.
The most important factor determining the success of the modified northern blotting protocol is the integrity of the RNA sample. RNAs with some amount of degradation may yield good classical northern blotting results, but this could potentially have significant impact on the modified northern blotting results. The modifications of RNA in different tissues or cell lines could also vary significantly. It is important to test the suitable RNA sample loads for different tissues before performing the final experiments.
As blotting procedures have been traditionally named after Dr. Southern and different geographical directions, we propose the name "northeastern" blotting for the current technique.

Dynamic and reversible RNA modifications have important roles in RNA homeostasis. Four decades ago, N6-methyladenosine (m6A) modifications were found to be abundant in eukaryotic transcriptomes1. They have diverse functions in messenger RNA (mRNA), ribosomal RNA (rRNA), small nucleolar RNA, transfer RNA, and microRNA2. The m6A modifications of mRNA influence their splicing3, translation4, degradation5, and localization2. Moreover, they affect ribosome biogenesis and microRNA function6. The evolutionary conservation of m6A modifications of RNA is noted in unicellular bacteria to multi-cellular humans7. Delineation of the roles of m6A modifications is currently under extensive exploration. The efforts are expected to provide new insights into transcription control. Recent studies reveal that other chemical modifications of mRNA8 also play critical roles in RNA metabolism. 
Circadian rhythms9, stem cell pluripotency10, triglyceride metabolism4, fibrosis11, obesity12, and major depression13 are a few examples of processes where m6A modifications are known to control outcomes. Many circadian clock gene transcripts have m6A sites14. Modulation of m6A methylase or demethylase elicits circadian period changes15. Mettl3, an m6A transferase, is a regulator for stem cell pluripotency. A deficiency of Mettl3 leads to early embryonic lethality and aberrant lineage priming at the post-implantation stage10. A deficiency of fat mass- and obesity-associated (FTO), an m6A demethylase, in adipocytes affects fatty acid mobilization and body weight through posttranscriptional regulation of Angptl44. These studies reveal that m6A not only controls mRNA processing, but also plays critical roles in embryological development and patho-physiology. The function of m6A modifications holds implications for therapeutic considerations in the future.
Several methods are available to measure m6A modifications of RNA16-18. Traditionally, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are used to study the distribution of m6A in several RNAs19,20. Mass spectrometry is a sensitive tool for the detection of m6A modifications in RNA. However, the RNA needs to be excised by RNase into short fragments before analysis by mass spectrometry21. Methyl-RNA immunoprecipitation and sequencing (m6A-Seq)22 immunoprecipitates fragmented RNAs with m6A-specific antibodies and performs parallel RNA sequencing. This method generates transcriptome-wide m6A landscapes. High-resolution mapping of m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) further maps m6A modifications at a single-nucleotide resolution23. Both methods provide details of m6A modification across the whole transcriptome, with specific genes' information. However, quantifications and standardization in both methods are difficult if experiments require the comparison of multiple conditions. Moreover, fragmentations of RNA for m6A-Seq alter the original RNA structure, which may affect native m6A levels. To detect global m6A modifications of RNA and their changes under different experimental conditions, we report a method that employs a modified northern blotting protocol. This method resolves RNA by molecular weight, using gel electrophoresis18. This procedure provides better standardization and quantifications for experiments that involve multiple conditions or samples. It also provides specific m6A modification information for different RNAs, whether rRNA, mRNA, or microRNA.

<table><tbody><tr><td>RNaseZap solution</td><td>Ambion</td><td>AM9782</td><td>Protocol 1.1</td></tr><tr><td>TRI Reagent solution</td><td>Ambion</td><td>AM9738</td><td>Protocol 1.2.1.1</td></tr><tr><td>1-Bromo-3-chloropropane (BCP)</td><td>Sigma</td><td>B9673</td><td>Protocol 1.2.1.3</td></tr><tr><td>Ethanol</td><td>JTbaker</td><td>8006</td><td>Protocol 1.2.1.3</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>I9516</td><td>Protocol 1.2.1.3</td></tr><tr><td>GenElute mRNA Miniprep Kit</td><td>Sigma</td><td>MRN70</td><td>Protocol 1.2.2.1</td></tr><tr><td>Glycogen</td><td>Ambion</td><td>AM9510</td><td>Protocol 1.2.2.2</td></tr><tr><td>Sodium acetate</td><td>Fluka</td><td>71183</td><td>Protocol 1.2.2.2</td></tr><tr><td>Nuclease-free water</td><td>Ambion</td><td>AM9930</td><td>Protocol 1.2.2.6</td></tr><tr><td>DEPC</td><td>Sigma</td><td>D5758</td><td>Protocol 2.1.1</td></tr><tr><td>Agarose</td><td>JT Baker</td><td>A426</td><td>Protocol 2.1.2</td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td>Protocol 2.1.3</td></tr><tr><td>37% formaldehyde Solution</td><td>Sigma</td><td>F8775</td><td>Protocol 2.1.3</td></tr><tr><td>EDTA, Disodium Salt</td><td>JT Baker</td><td>8993</td><td>Protocol 2.1.3 10x MOPS buffer</td></tr><tr><td>formamide</td><td>Sigma</td><td>F7503</td><td>Protocol 2.2.1</td></tr><tr><td>RNA Millennium Marker</td><td>Ambion</td><td>AM7150</td><td>Protocol 2.2.2</td></tr><tr><td>Ethidium Bromide</td><td>Amresco</td><td>X328</td><td>Protocol 2.2.5</td></tr><tr><td>Ficoll PM400</td><td>GE Healthcare</td><td>17-0300-10</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>114391</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Xylene Cyanol FF</td><td>Sigma</td><td>X4126</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Sodium chloride dihydrate</td><td>JT Baker</td><td>3624</td><td>Protocol 2.4.2 20x SSC buffer</td></tr><tr><td>Trisodium citrate</td><td>Sigma</td><td>S1804</td><td>Protocol 2.4.2 20x SSC buffer</td></tr><tr><td>Hybond Blotting Paper</td><td>GE Healthcare</td><td>RPN6101M</td><td>Protocol 2.4.4</td></tr><tr><td>GE Hybond-N+ membrane</td><td>GE Healthcare</td><td>RPN303B</td><td>Protocol 2.4.6</td></tr><tr><td>Stratalinker UV Crosslinker 2400</td><td>Stratagene</td><td>400075</td><td>Protocol 3.1.2</td></tr><tr><td>Gel Catcher 1500</td><td>ANT Technology</td><td>Gel Catcher 1500</td><td>Protocol 3.1.5</td></tr><tr><td>Anti-m&lt;sup&gt;6&lt;/sup&gt;A (N&lt;sup&gt;6&lt;/sup&gt;-methyladenosine)</td><td>Synaptic Systems</td><td>202003</td><td>Protocol 3.2.3</td></tr><tr><td>Amersham ECL Anti-Rabbit IgG, HRP-linkd whole Ab</td><td>GE Healthcare</td><td>NA934</td><td>Protocol 3.2.5</td></tr><tr><td>ChemiDoc MP System</td><td>BIO-RAD</td><td>1708280</td><td>Protocol 3.2.8</td></tr></tbody></table>

a method for measuring rna n6-methyladenosine modifications in cells and tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

Después de 14 días en la fase circadiana normal de luz-oscuridad, los ratones de tipo salvaje se colocaron en la oscuridad constante. Los ARN de hígado fueron muestreados a intervalos de 4 horas y estudiados con el norte de secante modificada. La metilación de rRNAs, mRNAs, y pequeños RNAs se detectaron claramente (Figura 2). La comparación entre diferentes momentos circadianos (CT) se puede calcular con precisión con el estándar de 18S rRNA. Hubo robusta oscilación circadiana de m 6 niveles A en ARNr, ARNm y ARN pequeños. Para evitar la interferencia ofrecida por ARNr, ARN poliadenilados se pueden purificar, como en el paso 1.2.2. Después de la purificación, el rRNA puede ser eliminado en gran medida para permitir una mejor visualización de los otros ARN (Figura 3). <img alt="Figura 1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html> g&gt; Figura 1: Montaje de la unidad de transferencia blot. Se muestra la unidad de transferencia capilar para secar el ARN a la membrana. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/54672/54672fig2.jpg" /> Figura 2: El ritmo circadiano de los m 6 niveles A en ratones C57BL / 6J. Esta figura muestra la m 6 Una mancha de ARN total de hígado de los ratones de tipo salvaje se sacrificó en el primer día de la fase oscura-oscura después de 14 días de una fase normal de luz-oscuridad a las 4 h intervalos. La cuantificación se realizó utilizando el m 6 A diferencia de densidad de imagen entre 18S y 28S rRNA. El m 6 Un abundancia se normalizó a la banda de 18S rRNA de la gel de formaldehído en la parte inferior.et = &quot;_ blank&quot;&gt; Haga clic aquí para ver una versión más grande de esta figura. <img alt="Figura 3" src="/files/ftp_upload/54672/54672fig3.jpg" /> Figura 3: Los m 6 borrones A con o sin rRNA. M representativos se muestran 6 A borrones de ARN total y ARN poliadenilado de los hígados de los ratones de tipo salvaje. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 1 </td><td colspan="3"> 10x tampón MOPS (pH 7,0, protegerlo de la luz) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> MOPS </td><td> 41.85 </td><td> gramo </td><td> 0,2 M </td></tr><tr><td></td><td colspan="3"> Acetato de sodio </td><td> 4.1 </td><td> gramo </td><td>0,05 M </td></tr><tr><td></td><td colspan="3"> EDTA, sal disódica </td><td> 3.7 </td><td> gramo </td><td> 0,01 M </td></tr><tr><td></td><td colspan="3"> agua DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Total </td><td> 1 </td><td> L </td><td></td></tr><tr><td></td><td colspan="3"> Se agita a temperatura ambiente </td><td></td><td></td><td></td></tr><tr><td> 2 </td><td colspan="3"> tampón SSC 20x (pH 7,0) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Cloruro de sodio </td><td> 175.3 </td><td> gramo </td><td> 3 M </td></tr><tr><td></td><td colspan="3"> Citrato de trisodio </td><td> 88.3 </td><td> gramo </td><td> 0,3 M </td></tr><tr><td></td><td colspan="3"> agua DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Total </td><td> 1 </td><td> L </td><td></td&gt; </tr><tr><td></td><td colspan="3"> Se agita a temperatura ambiente, a continuación, el autoclave </td><td></td><td></td><td></td></tr><tr><td> 3 </td><td colspan="3"> colorante guía </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10x tampón MOPS </td><td> 500 </td><td> l </td><td> 1x </td></tr><tr><td></td><td colspan="3"> Ficoll 400 </td><td> 0.75 </td><td> gramo </td><td> 15% </td></tr><tr><td></td><td colspan="3"> azul de bromofenol </td><td> 0.01 </td><td> gramo </td><td> 0,2% </td></tr><tr><td></td><td colspan="3"> xileno cianol </td><td> 0.01 </td><td> gramo </td><td> 0,2% </td></tr><tr><td></td><td colspan="3"> agua DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Total </td><td> 5 </td><td> ml </td><td></td></tr><tr><td></td><td colspan="3"> Almacenar a -20 ° C </td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> agua DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Diluir en relación 1: 1.000 de DEPC en ddH2O </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Se agita durante la noche a temperatura ambiente, a continuación, el autoclave </td><td></td><td></td><td></td></tr><tr><td> 5 </td><td colspan="3"> tampón de muestra (protegerlo de la luz) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10x tampón MOPS </td><td> 200 </td><td> l </td><td></td></tr><tr><td></td><td colspan="3"> 37% de formaldehído </td><td> 270 </td><td> l </td><td></td></tr><tr><td></td><td colspan="3"> formamida </td><td> 660 </td><td> l </td><td></td></tr><tr><td></td><td colspan="3"> Almacenar a -20 ° C </td><td></td><td></td><td></td></tr></tbody&gt; </table> Tabla 1: Los tampones y soluciones.

Watch this Scientific Journal Video about A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues at JoVE.com

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

Se describe un método de transferencia de Northern modificado para la medición de N 6 -methyladenosine (m 6 A) las modificaciones en el ARN. El método actual puede detectar modificaciones en diversos ARN y controles en virtud de diversos diseños experimentales.

Un método para medir el ARN<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine Modificaciones en células y tejidos

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

a-method-for-measuring-rna-n6-methyladenosine-modifications-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

10.8K vistas.  Chang Gung Memorial Hospital, Chang Gung University College of Medicine.  Se describe un método de transferencia de Northern modificado para la medición de N 6 -methyladenosine (m 6 A) las modificaciones en el ARN. El método actual puede detectar modificaciones en diversos ARN y controles en virtud de diversos diseños experimentales. 

Video: A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

Un ensayo In Vitro para detectar actividad de transferasa de Isopentenyl tRNA

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Investigación

Bioquímica

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

Ensayo libre de anticuerpos para el análisis de actividad de la metiltransferasa de ARN

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

Genética

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

Mejora de ARN pequeño-seq: Menos sesgo y mejor detección de 2'-O-Metil RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

The methylated RNA immunoprecipitation assay is an antibody-based method used to enrich for methylated RNA fragments. Coupled with deep sequencing, it leads to the identification of transcripts carrying the m5C modification.

Ensayo de inmunoprecipitación de ARN metilado para estudiar la modificación m5C en Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

<meta charset="utf8"></head><body>2D-HELS MS Seq: Un método general basado en LC-MS para la secuenciación directa y de novo de mezclas de ARN con diferentes modificaciones de nucleótidos

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

2D-HELS MS Seq: Un método general basado en LC-MS para la secuenciación directa y de novo de mezclas de ARN con diferentes modificaciones de nucleótidos

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Explorando el metiloma de arginina mediante espectroscopia de resonancia magnética nuclear

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Investigación sobre el cáncer

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

Preparación de muestras de plantas para la medición del contenido de nucleósidos/nucleótidos con una HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

Análisis dependiente de DNAzyme de rRNA 2'-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

Biología del Desarrollo

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described&#8212;single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)&#8212;to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

Caracterización de modificaciones de ARN en neuronas individuales mediante espectrometría de masas

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...

Un método para medir el ARN

Resumen

Explorar más videos

Capítulos en este video

Un ensayo In Vitro para detectar actividad de transferasa de Isopentenyl tRNA

Ensayo libre de anticuerpos para el análisis de actividad de la metiltransferasa de ARN

Mejora de ARN pequeño-seq: Menos sesgo y mejor detección de 2'-O-Metil RNAs

Ensayo de inmunoprecipitación de ARN metilado para estudiar la modificación m⁵C en Arabidopsis

2D-HELS MS Seq: Un método general basado en LC-MS para la secuenciación directa y de novo de mezclas de ARN con diferentes modificaciones de nucleótidos

2D-HELS MS Seq: Un método general basado en LC-MS para la secuenciación directa y de novo de mezclas de ARN con diferentes modificaciones de nucleótidos

Explorando el metiloma de arginina mediante espectroscopia de resonancia magnética nuclear

Preparación de muestras de plantas para la medición del contenido de nucleósidos/nucleótidos con una HPLC-MS/MS

Análisis dependiente de DNAzyme de rRNA 2'-O-Methylation

Caracterización de modificaciones de ARN en neuronas individuales mediante espectrometría de masas