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).

HINWEIS: Die Gesamt - RNA m 6 A Ebene umfasst rRNA, mRNA, und andere kleine RNAs. Da ribosomale RNA hat reichlich m 6 A Änderungen, müssen die Messung von m 6 A - Levels , diese Tatsache zu berücksichtigen. 1. RNA-Isolierung <ol><li> Mit Hilfe der Ribonuklease Dekontaminationslösung (gesprüht auf Papiertücher), reinigen Sie die Pipetten und Labortisch Oberflächen. Nasse Papiertücher mit Nuklease-freies Wasser und wischen Sie Pipetten und Labortisch taucht wieder auf. </li><li> RNA-Extraktion <ol><li> Gesamt-RNA-Isolation <ol><li> Mit einem Overhead - Rührer, homogenisieren 50-100 mg Gewebe oder 5-10 x 10 6 Zellen in 1 ml RNA Isolationslösung bei 4 ° C gehalten. HINWEIS: Weitere Gewebe (100-150 mg) für Fettgewebe Proben von Mäusen erforderlich wegen der niedrigeren RNA-Konzentrationen darin. Proben aus Mäuse Herz, Leber, Skelettmuskel, Lunge, Gehirn stammen und Makrophagen zuvor 4 eingesetzt wurden. </li><li> Imcubate die Probe Homogenate für 5 Minuten bei Raumtemperatur. </li><li> Füge 100 &amp; mgr; l 1-Brom-3-chlorpropan (BCP) pro 1 ml Probe Homogenat. Schütteln der Röhrchen kräftig für 15 s und verfahren Gesamt - RNA zu isolieren , gemäß den Anweisungen des Herstellers 24. </li></ol></li><li> Die polyadenylierte mRNA Purification von isolierten Gesamt-RNA <ol><li> Reinige polyadenylierte mRNA unter Verwendung von erhältlichen Kits oder Protokolle. <ol><li> Schütteln gründlich resuspendieren jeder der folgenden Lösungen: 2x Bindungslösung, Oligo (dT) Polystyrol-Kügelchen und Waschlösung. Sicherzustellen, dass das Oligo (dT) -Kügelchen erwärme auf Raumtemperatur vor dem Gebrauch. </li><li> Übertragung von 120 &amp; mgr; l Elutionslösung pro Zubereitung in ein Rohr und Wärme auf 70 ° C in einem Heizblock. </li><li> Pipette bis zu 500 ug Gesamt-RNA in ein Mikrozentrifugenröhrchen. Mit nukleasefreiem Wasser, stellen Sie die Lautstärke auf 250 &amp; mgr; l. </li><li> In 250 ul 2x Bindungslösung auf die Gesamt-RNA solution und Wirbel kurz den Inhalt zu mischen. </li><li> In 15 ul Oligo (dT) Perlen und Wirbel gründlich zu mischen. </li><li> Inkubieren der Mischung bei 70 ° C für 3 min. </li><li> Entfernen Sie die Probe aus dem Heizblock und lassen Sie es 10 Minuten lang bei Raumtemperatur stehen. </li><li> Zentrifuge bei 15.000 g für 2 min. </li><li> Entfernen Sie vorsichtig den Überstand, hinter etwa 50 &amp; mgr; l zu verlassen. </li><li> In 500 ul Waschlösung zum Resuspendieren des Pellets durch Pipettieren. </li><li> Pipette, um die Suspension in einen Spinfilter / Sammelrohr-Set. </li><li> Zentrifuge bei 15.000 g für 2 min. Entfernen Sie die Säule aus Sammelröhrchen und entsorgen Sie die Durchfluss. Bringen Sie die Spalte an die Sammelröhrchen. </li><li> Pipette 500 ul Waschlösung auf den Spinfilter. </li><li> Zentrifuge bei 15.000 x g für 2 min. </li><li> Übertragen Sie die Spinfilter in ein frisches Sammelröhrchen. </li><li> Pipettieren von 50 &amp; mgr; l Elutionslösung erhitztbis 70 ° C auf das Zentrum des Spinfilters. </li><li> Inkubieren für 2-5 min bei 70 ° C. Zentrifuge bei 15.000 × g für 1 min. </li><li> Pipettieren zusätzlich 50 &amp; mgr; l Elutionslösung auf 70 ° C auf das Zentrum des Spinfilters. </li><li> Wiederholen Sie Schritt 1.2.2.1.18. </li></ol></li><li> Je 100 &amp; mgr; g / mL Glykogen, 0,1 Volumen von 3 M Natriumacetat-Puffer (pH 5,2) und 2,5 Volumina absolutem Ethanol. Man fällt über Nacht bei -80 ° C. </li><li> Zentrifuge bei 15.000 xg für 25 min bei 4 ° C. Überstand verwerfen. </li><li> Waschen Sie das Pellet mit 1 ml 75% Ethanol. Zentrifuge bei 15.000 xg für 15 min bei 4 ° C. Sie vorsichtig das Ethanol zu entfernen. </li><li> Trockene Luft für 3-5 min. </li><li> Fügen Sie 10 ul Nuklease-freies Wasser, um das Pellet aufzulösen. </li><li> Lagern Sie bei -70 ° C. </li></ol></li></ol></li><li> RNA Qualifizierung und Quantifizierung <ol><li> Verwendung eines Spektrophotometers, bestimmen die RNA-Konzentration durch NotIng der Absorption bei 260 nm und 280 nm. Die A 260 / A 280 Verhältnis sollte 1,8-2,2 sein. </li><li> Schauen Sie sich die RNA - Probe Qualität unter Verwendung von 0,8% Agarose - Gelelektrophorese 25. HINWEIS: Die intakte eukaryotischen Gesamt-RNA intensiv 28S und 18S rRNA-Banden zeigen. Das Verhältnis von 28S rRNA 18S gegen Bandenintensität für eine gute RNA-Präparation ist ~ 2. </li></ol></li></ol> 2. Gel-Elektrophorese und Übertragung HINWEIS: Die Puffervorbereitungsprotokollen sind in Tabelle 1. <ol><li> Formaldehyd-Gel-Vorbereitung (1%) <ol><li> Spülen Sie alle Elektrophoreseapparaturen mit Diethylpyrocarbonat (DEPC) Wasser. </li><li> Schmelze 2,5 g Agarose in 215 ml DEPC Wasser vollständig in einem Mikrowellenofen. </li><li> Hinzufügen 12,5 ml 10x 3- (N- Morpholino) propansulfonsäure (MOPS) -Puffer und 22,5 ml 37% Formaldehyd. </li><li> Gießen Sie es in die Elektrophorese-Gerät mit einer 1,5 mm dicken Kamm. </li><li> Beseitigen Sie Blasen oder Push them zu den Kanten des Gels mit einem sauberen comb. </li><li> Lassen Sie das Gel bei Raumtemperatur zu verfestigen. </li></ol></li><li> Probenvorbereitung <ol><li> Mischen 1-10 ug Gesamt-RNA und 11,3 &amp; mgr; l Probenpuffer. </li><li> Mix 2 ug der RNA-Marker (1 &amp; mgr; g / &amp; mgr; l) mit 11,3 &amp; mgr; l des Probenpuffers. </li><li> In nukleasefreiem Wasser zu den Proben von 2.2.1 und 2.2.2 bis zu einem Gesamtvolumen von 16 &amp; mgr; l. </li><li> Hitze bei 60 ° C für 5 min, und dann auf Eis kühlen. </li><li> Mischungs 16 &amp; mgr; l der Probe aus 2.2.3 mit 4 ul Tracking-Farbstoff, welche 0,1 &amp; mgr; g enthält / &amp; mgr; l Ethidiumbromid auf Eis. ACHTUNG: Ethidiumbromid ist möglicherweise teratogen und giftig beim Einatmen. Lesen Sie Ethidiumbromid Sicherheitsdatenblatt. </li></ol></li><li> Elektrophorese <ol><li> In 200 ml 10x MOPS bis 1.800 ml ddH 2 O - Puffer mit einem 1x MOPS Laufpuffer zu machen. </li><li> Verwenden Sie die 1x MOPS Laufpuffer die Vertiefungen des Gels zu spülen. </li><li> Pre-run ter Gel bei 20 V für 5 min. </li><li> Laden die RNA-Proben in die Vertiefungen des Gels. </li><li> Mit Folie abdecken Lichtexposition zu vermeiden. Führen Sie bei 35 V für etwa 17 Stunden (über Nacht) </li><li> Untersuchen und fotografieren Sie das Gel unter UV-Licht. </li></ol></li><li> Übertragung <ol><li> Schneiden Sie das Gel den ungenutzten Teil zu entfernen. </li><li> Bereiten 500 ml 10x SSC-Puffer (250 ml 20x SSC-Puffer und 250 ml DEPC-Wasser). </li><li> Waschen Sie das Gel zweimal mit 10x SSC-Puffer für 20 min mit 50 Umdrehungen pro Minute schütteln. </li><li> Cut 1 Filterpapierblatt groß genug , um als Docht Papier zu dienen, die Transferpuffer aus der Übertragungsschale (Abbildung 1) aufnimmt. </li><li> Schneiden 4 Stück Filterpapier der gleichen Größe wie das Gel. </li><li> Geschnitten 1 Blatt positiv geladene Nylonmembran auf die gleiche Größe wie das Gel. </li><li> Markieren eine Kerbe auf dem Gel und die Membran als eine Markierung, um die richtige Ausrichtung zu gewährleisten. </li><li> Einweichen der Membran in 2 × SSC-Puffer für 15 min. </li><li> Gießen Sie 500 ml of 10x SSC-Puffer in die Transferschale. </li><li> Legen Sie das große Filterpapierbogen über der Glasplatte und benetzt das Filterpapier mit Transferpuffer. </li><li> Ausrollen alle Blasen mit einer Pipette. </li><li> Soak 2 Stück vorgeschnittene Filterpapier in Transferpuffer und legen sie auf die Mitte des Filterpapiers aus Schritt 2.4.5. Entfernen Sie alle Blasen. </li><li> Legen Sie das Gel von oben nach unten auf dem Filterpapier. </li><li> Legen Sie die Membran auf der Oberseite des Gels. Ausrichten der Kerben des Gels und der Membran. </li><li> Platz 2 Stücke von vorgeschnittenen Filterpapier auf der Oberseite der Membran und benetzt das Filterpapier mit Transferpuffer. </li><li> Entfernen Sie alle Blasen zwischen den Schichten. </li><li> Tragen Sie Plastikfolie um das Gel, um sicherzustellen, dass die Handtücher genießen nur Puffer durch das Gel vorbei und die Membran. </li><li> Stapeln Sie die Papiertücher auf dem Filterpapier. </li><li> Legen Sie eine überdimensionale Glasplatte auf der Oberseite der Handtücher. </li><li> Legen Sie ein Gewicht auf der Oberseite des Stapels. </li><li>Halten über Nacht die RNA zu ermöglichen, aus dem Gel auf die Membran zu übertragen. </li></ol></li></ol> 3. Nachweis von N 6 -methyladenosine <ol><li> Membran-Crosslinking <ol><li> Halten Sie die Membran feucht in 2x SSC-Puffer. </li><li> Platz 2 Blätter Filterpapier eingetaucht mit 10x SSC-Puffer in den UV-Vernetzer. </li><li> Platzieren Sie die Membran auf der Oberseite des Filterpapiers, mit dem RNA-adsorbierten Seite nach oben. </li><li> Wählen Sie &quot;autocrosslink Modus&quot; (120.000 &amp; mgr; J) und drücken Sie die Schaltfläche &quot;Start&quot; die Bestrahlung zu initiieren. </li><li> Untersuche die Membran und das Gel mit einem UV Gelbild Fängers die RNA-Transfer von dem Gel auf die Membran zu bestätigen. </li></ol></li><li> Immunoblotting <ol><li> Blockieren die Membran in 5% fettfreier Milch in Tris-gepufferter Kochsalzlösung mit Tween 20 (TBST) Puffer bei Raumtemperatur für 1 Stunde. </li><li> Waschen dreimal mit TBST-Puffer für 15 min. </li><li> Inkubieren Sie die Membran über Nacht in m 6 A (&lt;em&gt; N 6 -methyladenosine) Antikörperlösung (1: 1000 in 5% fettfreier Milch TBST - Puffer) bei 4 ° C. </li><li> Waschen Sie die Membran dreimal mit TBST-Puffer für 15 min. </li><li> Inkubieren der Membran in dem HRP-konjugierter Esel-Anti-Kaninchen-Antikörperlösung (1: 2000 in 5% fettfreier Milch TBST-Puffer) für 1 h bei Raumtemperatur. </li><li> Waschen Sie die Membran dreimal mit TBST-Puffer für 15 min. </li><li> Wenden Sie den verstärkten chemolumineszenten Substrat (0,125 ml pro cm 2 Membran). </li><li> Erfassen die Chemilumineszenz mit den optimalen Einstellungen des Digital Imager nach den Anweisungen des Herstellers 26. </li></ol></li><li> m 6 A Quantifizierung <ol><li> Messen Sie die relative m 6 A Chemilumineszenzintensität mit der ImageJ Software. <ol><li> Unter dem ImageJ Software Wählen Sie im Menü &quot;Datei&quot; Option, um die zugehörigen Datei zu öffnen. </li><li> Wählen Sie das &quot;Rechteck&quot; Werkzeug aus ImageJ und einen Rahmen ziehen umdie Signale. </li><li> Wenn ribosomale RNA nicht im Mittelpunkt der Experimente ist, vermeiden Sie die 18S und 28S ribosomale RNA m 6 A Bänder für die Berechnung. </li><li> Klicken Sie auf &quot;Befehl&quot; und &quot;1&quot; die gewählte Spur zu bestätigen. </li><li> Drücken Sie die &quot;Command&quot; und &quot;3&quot; das ausgewählte Grundstück zu zeigen. </li><li> Klicken Sie auf den &quot;Straight&quot; Werkzeug und Linien zeichnen den segmentierten Bereich zu trennen. </li><li> Klicken Sie auf die &quot;Wand&quot; Werkzeug, um die Messungen aufzuzeichnen. </li><li> Exportieren Sie die Daten. </li><li> Normalisieren die Niveaus von m 6 A mit der 18S ribosomale RNA - Bande aus dem Ethidiumbromidfärbung. </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>

Die Autoren haben nichts zu offenbaren.

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.

Nach 14 Tagen im normalen Licht-Dunkel-zirkadianen Phase, die Wildtyp-Mäuse wurden in konstanter Dunkelheit platziert. Die RNAs aus der Leber wurden bei 4 Stunden Intervallen abgetastet und mit modifiziertem Northern-Blotting untersucht. Die Methylierung von rRNAs, mRNAs und kleine RNAs wurden eindeutig nachgewiesen (Figur 2). Der Vergleich zwischen den verschiedenen Tageszeiten (CT) kann genau mit der 18S rRNA-Standard berechnet werden. Es gab robuste zirkadiane Oszillation von m 6 A - Spiegel in rRNA, mRNA und kleinen RNAs. Um die Störungen zu vermeiden, indem rRNA dargebotene kann polyadenylierte RNA gereinigt werden, wie es in Schritt 1.2.2. Nach der Reinigung kann die rRNA weitgehend eliminiert zur besseren Visualisierung der anderen RNAs (Figur 3) zu ermöglichen. <img alt="Abbildung 1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html> g&gt; Abbildung 1: Die Blot-Transfereinheit zusammenbauen. Die kapillare Übertragungseinheit für die RNA an die Membran-Blotting gezeigt. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/54672/54672fig2.jpg" /> Abbildung 2: Circadian Rhythmus der m 6 A - Spiegel in C57BL / 6J - Mäusen. Diese Abbildung zeigt die m 6 A - Blot von Gesamt - RNA aus der Leber von Wildtyp - Mäusen am ersten Tag des Dunkeldunkelphase nach 14 Tagen einer normalen Hell-Dunkel - Phase bei 4 h Intervallen geopfert. Die Quantifizierung wurde mit der m getan 6 A Bilddichteunterschied zwischen 18S und 28S rRNA. Die m 6 A Fluss wurde am Boden mit dem 18S - rRNA - Band aus dem Formaldehyd - Gel normalisiert.et = &quot;_ blank&quot;&gt; Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen. <img alt="Figur 3" src="/files/ftp_upload/54672/54672fig3.jpg" /> Abbildung 3: Die m 6 A Blots mit oder ohne rRNA. Representative m 6 A - Blots von Gesamt - RNA und polyadenylierte RNA aus der Leber von Wildtyp - Mäusen gezeigt. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</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 MOPS-Puffer (pH 7,0, vor Licht schützen) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> MOPS </td><td> 41,85 </td><td> G </td><td> 0,2 M </td></tr><tr><td></td><td colspan="3"> Natriumacetat </td><td> 4.1 </td><td> G </td><td>0,05 M </td></tr><tr><td></td><td colspan="3"> EDTA, Dinatriumsalz </td><td> 3.7 </td><td> G </td><td> 0,01 M </td></tr><tr><td></td><td colspan="3"> DEPC Wasser </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Gesamt </td><td> 1 </td><td> L </td><td></td></tr><tr><td></td><td colspan="3"> Rühre bei Raumtemperatur </td><td></td><td></td><td></td></tr><tr><td> 2 </td><td colspan="3"> 20x SSC-Puffer (pH 7,0) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Kochsalz </td><td> 175,3 </td><td> G </td><td> 3 M </td></tr><tr><td></td><td colspan="3"> Trinatriumcitrat </td><td> 88.3 </td><td> G </td><td> 0,3 M </td></tr><tr><td></td><td colspan="3"> DEPC Wasser </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Gesamt </td><td> 1 </td><td> L </td><td></td&gt; </tr><tr><td></td><td colspan="3"> Rühre bei Raumtemperatur, dann autoklaviert </td><td></td><td></td><td></td></tr><tr><td> 3 </td><td colspan="3"> Tracking-Dye </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10x MOPS-Puffer </td><td> 500 </td><td> ul </td><td> 1x </td></tr><tr><td></td><td colspan="3"> Ficoll 400 </td><td> 0,75 </td><td> G </td><td> 15% </td></tr><tr><td></td><td colspan="3"> Bromphenolblau </td><td> 0,01 </td><td> G </td><td> 0,2% </td></tr><tr><td></td><td colspan="3"> Xylencyanol </td><td> 0,01 </td><td> G </td><td> 0,2% </td></tr><tr><td></td><td colspan="3"> DEPC Wasser </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Gesamt </td><td> 5 </td><td> ml </td><td></td></tr><tr><td></td><td colspan="3"> Lagerung bei -20 ° C </td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> DEPC Wasser </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Verdünnen Verhältnis bei 1: 1000 von DEPC in ddH 2 O </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Rühre über Nacht bei Raumtemperatur, dann autoklaviert </td><td></td><td></td><td></td></tr><tr><td> 5 </td><td colspan="3"> Probenpuffer (vor Licht schützen) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10x MOPS-Puffer </td><td> 200 </td><td> ul </td><td></td></tr><tr><td></td><td colspan="3"> 37% Formaldehyd </td><td> 270 </td><td> ul </td><td></td></tr><tr><td></td><td colspan="3"> Formamid </td><td> 660 </td><td> ul </td><td></td></tr><tr><td></td><td colspan="3"> Lagerung bei -20 ° C </td><td></td><td></td><td></td></tr></tbody&gt; </table> Tabelle 1: Puffer und Lösungen.

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

Modifiziertes Northern - Blotting - Verfahren für N 6 -methyladenosine (m 6 A) Änderungen in RNA Messung beschrieben. Die aktuelle Methode können Änderungen in diversen RNAs und Kontrollen unter verschiedenen experimentellen Designs erkennen.

Verfahren zum Messen von RNA<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine Änderungen in Zellen und Geweben

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 Aufrufe.  Chang Gung Memorial Hospital, Chang Gung University College of Medicine.  Modifiziertes Northern - Blotting - Verfahren für N 6 -methyladenosine (m 6 A) Änderungen in RNA Messung beschrieben. Die aktuelle Methode können Änderungen in diversen RNAs und Kontrollen unter verschiedenen experimentellen Designs erkennen. 

Serie: 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.

Ein In vitro- Test zur Erkennung der tRNA-Isopentenyl-Transferase-Aktivität

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

Forschung

Biochemie

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.

Antikörperfreier Assay für rna-Methyltransferase-Aktivitätsanalyse

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

Genetik

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.

Verbesserung der kleinen RNA-Seq: Weniger Voreingenommenheit und bessere Detektion von 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 ...

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.

Methylierte RNA-Immunpräzipitierung Assay zur Studie m5C Modifikation bei 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: Eine allgemeine LC-MS-basierte Methode zur direkten und de novo Sequenzierung von RNA-Gemischen mit unterschiedlichen Nukleotidmodifikationen

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: Eine allgemeine LC-MS-basierte Methode zur direkten und de novo Sequenzierung von RNA-Gemischen mit unterschiedlichen Nukleotidmodifikationen

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.

Erforschung des Arginin-Methyloms durch Kernspinresonanzspektroskopie

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

Krebsforschung

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.

Pflanzenprobenvorbereitung zur Messung des Nukleosid-/Nukleotidgehalts mit EINER 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.

DNAzyme-abhängige Analyse der rRNA 2'-O-Methylierung

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 ...

Entwicklungsbiologie

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.

Charakterisierung von RNA-Modifikationen in einzelnen Neuronen mittels Massenspektrometrie

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

Verfahren zum Messen von RNA

Zusammenfassung

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