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: L&#39;RNA totale m 6 A livello comprende rRNA, mRNA, e altri piccoli RNA. Dal momento che l&#39;RNA ribosomiale è abbondante m 6 modifiche A, la misura di m 6 livelli A dovranno prendere in considerazione questo fatto. Isolamento 1. RNA <ol><li> Utilizzando la soluzione di decontaminazione ribonucleasi (spruzzato su tovaglioli di carta), pulire le pipette e le superfici banco di laboratorio. asciugamani di carta bagnati con acqua priva di nucleasi e pulire le pipette e le superfici banco di laboratorio di nuovo. </li><li> RNA Estrazione <ol><li> Isolamento RNA totale <ol><li> Utilizzando un agitatore, mescolare 50-100 mg di tessuto o 5-10 x 10 6 cellule in 1 ml di soluzione di isolamento dell&#39;RNA mantenuti a 4 ° C. NOTA: più tessuto (100-150 mg) può essere richiesto per i campioni di tessuto adiposo di topi a causa delle concentrazioni di RNA inferiori in esso. I campioni provengono da cuore del mouse, fegato, muscolo scheletrico, polmone, cervello e macrofagi sono stati impiegati in precedenza 4. </li><li> Incubate omogenati di esempio per 5 min a temperatura ambiente. </li><li> Aggiungere 100 ml di 1-bromo-3-cloropropano (BCP) per 1 ml di omogenato del campione. Agitare le provette con forza per 15 s e procedere per isolare l&#39;RNA totale secondo le istruzioni 24 del produttore. </li></ol></li><li> Poliadenilato mRNA Purificazione da RNA totale isolato <ol><li> Purificare mRNA poliadenilato utilizzando kit o protocolli disponibili. <ol><li> Agitare accuratamente per risospendere ciascuna delle seguenti soluzioni: soluzione vincolante, oligo (dT) perle di polistirene, e soluzione di lavaggio 2x. Assicurarsi che il oligo (dT) perline calda a temperatura ambiente prima dell&#39;uso. </li><li> Trasferire 120 ml di soluzione di eluizione per la preparazione in un tubo e si riscalda a 70 ° C su una piastra riscaldante. </li><li> Pipetta fino a 500 mg di RNA totale in una provetta. Con l&#39;acqua priva di nucleasi, regolare il volume a 250 ml. </li><li> Aggiungere 250 ml di soluzione vincolante 2x alla RNA totale cosìluzione e brevemente vortex per mescolare il contenuto. </li><li> Aggiungere 15 ml di oligo (dt) perline e vortex accuratamente per mescolare. </li><li> Incubare la miscela a 70 ° C per 3 min. </li><li> Rimuovere il campione dal blocco di riscaldamento e lasciar riposare a temperatura ambiente per 10 min. </li><li> Centrifugare a 15.000 xg per 2 min. </li><li> Rimuovere con attenzione il surnatante, lasciando dietro di sé circa 50 ml. </li><li> Aggiungere 500 ml di soluzione di lavaggio per risospendere il pellet pipettando. </li><li> Pipettare la sospensione in un set di tubi filtro rotativo / collezione. </li><li> Centrifugare a 15.000 xg per 2 min. Rimuovere la colonna dal tubo di raccolta e scartare il flow-through. Riportare la colonna a tubo di raccolta. </li><li> Pipettare 500 ml di soluzione di lavaggio sul filtro rotativo. </li><li> Centrifugare a 15.000 x g per 2 minuti. </li><li> Trasferire il filtro rotativo in un tubo di raccolta fresca. </li><li> Pipettare 50 ml di soluzione di eluizione riscaldataa 70 ° C sul centro del filtro rotativo. </li><li> Incubare per 2-5 minuti a 70 ° C. Centrifugare a 15.000 xg per 1 min. </li><li> Pipettare un ulteriore 50 ml di soluzione di eluizione riscaldata a 70 ° C sul centro del filtro rotativo. </li><li> Ripetere il passo 1.2.2.1.18. </li></ol></li><li> Aggiungere 100 mg glicogeno / mL, 0,1 volumi di tampone di 3 M acetato di sodio (pH 5,2) e 2,5 volumi di etanolo assoluto. Precipitato durante la notte a -80 ° C. </li><li> Centrifugare a 15.000 xg per 25 min a 4 ° C. Eliminare il surnatante. </li><li> Lavare il pellet con 1 ml di etanolo al 75%. Centrifugare a 15.000 xg per 15 min a 4 ° C. Rimuovere con attenzione l&#39;etanolo. </li><li> A secco in aria per 3-5 minuti. </li><li> Aggiungere 10 ml di acqua priva di nucleasi per dissolvere il pellet. </li><li> Conservare a -70 ° C. </li></ol></li></ol></li><li> RNA qualificazione e quantificazione <ol><li> Utilizzando uno spettrofotometro, determinare la concentrazione di RNA da noting l&#39;assorbanza a 260 nm e 280 nm. Il rapporto A 260 / A 280 dovrebbe essere 1,8-2,2. </li><li> Controllare la qualità del campione di RNA utilizzando 0,8% gel elettroforesi 25. NOTA: L&#39;RNA totale eucariotica intatto dovrebbe mostrare intense bande 28S e 18S rRNA. Il rapporto di 28S contro intensità banda 18S rRNA per una buona preparazione RNA è ~ 2. </li></ol></li></ol> 2. elettroforesi su gel e di trasferimento NOTA: I protocolli di preparazione del buffer sono riportati nella tabella 1. <ol><li> Formaldeide Gel di Preparazione (1%) <ol><li> Risciacquare tutte le apparecchiature elettroforesi con dietilpirocarbonato acqua (DEPC). </li><li> Sciogliere 2.5 g di agarosio in 215 mL di acqua DEPC completamente in un forno a microonde. </li><li> Aggiungere 12,5 ml di 10x 3- (N-morfolino) propanosulfonico (MOPS) buffer e 22,5 ml di formaldeide al 37%. </li><li> Versare il tutto nell&#39;apparecchio elettroforesi con un pettine 1,5 mm di spessore. </li><li> Eliminare le bolle o spingere torlo per i bordi del gel con un pettine pulito. </li><li> Lasciare il gel solidificare a temperatura ambiente. </li></ol></li><li> Preparazione del campione <ol><li> Mescolare 1-10 mg di RNA totale e 11,3 ml di tampone campione. </li><li> Mescolare 2 ug del marcatore RNA (1 mg / mL) con 11.3 ml di tampone campione. </li><li> Aggiungere acqua priva di nucleasi ai campioni da 2.2.1 e 2.2.2 ad un volume totale di 16 microlitri. </li><li> Riscaldare a 60 ° C per 5 minuti, e poi raffreddare su ghiaccio. </li><li> Mescolare 16 microlitri del campione da 2.2.3 con 4 ml di colorante inseguimento, che contiene 0,1 mg / mL di bromuro di etidio in ghiaccio. ATTENZIONE: il bromuro di etidio è forse teratogeno e tossico se inalato. Leggi bromuro di etidio scheda di sicurezza. </li></ol></li><li> elettroforesi <ol><li> Aggiungere 200 ml di 10x tampone MOPS a 1.800 ml di DDH 2 O per fare un tampone di corsa 1x MOPS. </li><li> Utilizzare il tampone di corsa 1x MOPS per sciacquare i pozzetti del gel. </li><li> Pre-run tegli gel a 20 V per 5 min. </li><li> Caricare i campioni di RNA nei pozzetti del gel. </li><li> Coprire con un foglio per evitare l&#39;esposizione alla luce. Eseguire a 35 V per circa 17 ore (durante la notte) </li><li> Esaminare e fotografare il gel sotto una luce UV. </li></ol></li><li> Trasferimento <ol><li> Tagliare il gel per rimuovere la parte non utilizzata. </li><li> Preparare 500 ml di 10x tampone SSC (250 mL di 20x tampone SSC e 250 mL di acqua DEPC). </li><li> Lavare il gel due volte con 10x tampone SSC per 20 minuti con agitazione a 50 giri al minuto. </li><li> Tagliare 1 filtro foglio di carta grande abbastanza per servire come una carta stoppino, che assorbe buffer di trasferimento dalla sezione Trasferimento (Figura 1). </li><li> Tagliare 4 pezzi di carta da filtro per la stessa dimensione del gel. </li><li> Tagliare 1 foglio di membrana di nylon caricata positivamente alla stessa dimensione come il gel. </li><li> Tracciare una tacca sul gel e la membrana come marcatore per assicurare il corretto orientamento. </li><li> Immergere la membrana in tampone SSC 2x per 15 min. </li><li> Versare 500 ml of 10x SSC tampone nel cassetto di trasferimento. </li><li> Disporre il grande foglio di carta filtrante attraverso la piastra di vetro e bagnare la carta da filtro con il tampone di trasferimento. </li><li> Stendete le bolle con una pipetta. </li><li> Immergere 2 pezzi di carta da filtro pre-tagliati in buffer di trasferimento e metterli al centro della carta da filtro dal punto 2.4.5. Rimuovere eventuali bolle. </li><li> Posare il gel superiore rivolto verso il basso sulla carta da filtro. </li><li> Disporre la membrana sulla superficie del gel. Allineare le tacche del gel e la membrana. </li><li> Mettere 2 pezzi di carta filtro pre-taglio sulla parte superiore della membrana e bagnare la carta da filtro con il tampone di trasferimento. </li><li> Rimuovere eventuali bolle tra gli strati. </li><li> Applicare pellicola intorno al gel di garantire che gli asciugamani assorbono solo fino tampone passa attraverso il gel e la membrana. </li><li> Stack i tovaglioli di carta sulla parte superiore della carta da filtro. </li><li> Inserire un lastra di vetro di grandi dimensioni sulla parte superiore degli asciugamani. </li><li> Collocare un peso in cima alla pila. </li><li>Mantenere notte per consentire l&#39;RNA di trasferire dal gel alla membrana. </li></ol></li></ol> 3. Rilevamento di N 6 -methyladenosine <ol><li> membrana reticolazione <ol><li> Mantenere la membrana umida nel tampone 2x SSC. </li><li> Mettere 2 fogli di carta da filtro immersa con 10x SSC tampone in reticolante UV. </li><li> Posizionare la membrana sulla parte superiore della carta da filtro, con il lato di RNA-adsorbito rivolto verso l&#39;alto. </li><li> Selezionare &quot;modalità autocrosslink&quot; (120.000 μJ) e premere il pulsante &quot;Start&quot; per avviare l&#39;irradiazione. </li><li> Esaminate la membrana ed il gel con UV immagine Gel collettore per confermare il trasferimento RNA dal gel alla membrana. </li></ol></li><li> immunoblotting <ol><li> Bloccare la membrana in latte scremato 5% in soluzione salina tamponata con Tris con Tween 20 (TBST) tampone a temperatura ambiente per 1 ora. </li><li> Lavare tre volte con tampone TBST per 15 min. </li><li> Incubare la membrana durante la notte in m 6 A (&lt;em&gt; soluzione N 6 -methyladenosine) anticorpo (1: 1.000 in 5% senza grassi del latte tampone TBST) a 4 ° C. </li><li> Lavare la membrana tre volte con tampone TBST per 15 min. </li><li> Incubare la membrana nella soluzione HRP-coniugato asino anti-coniglio anticorpo (1: 2.000 in tampone latte TBST 5% non grassa) per 1 ora a temperatura ambiente. </li><li> Lavare la membrana tre volte con tampone TBST per 15 min. </li><li> Applicare il substrato chemiluminescente potenziato (0,125 ml per cm 2 di membrana). </li><li> Cattura il chemiluminescenza con le impostazioni ottimali del imager digitale, secondo le istruzioni del produttore 26. </li></ol></li><li> m 6 una quantificazione <ol><li> Misurare la relativa m 6 A intensità chemiluminescente utilizzando il software ImageJ. <ol><li> Sotto il menu del software ImageJ, selezionare l&#39;opzione &quot;File&quot; per aprire il file pertinente. </li><li> Selezionare lo strumento &quot;Rettangolo&quot; da ImageJ e disegnare una cornice intornoi segnali. </li><li> Se RNA ribosomiale non è il focus degli esperimenti, evitare le 18S e 28S RNA ribosomiale m 6 fasce A per il calcolo. </li><li> Fai clic su &quot;Comando&quot; e &quot;1&quot; per confermare la corsia selezionato. </li><li> Premere il tasto &quot;Comando&quot; e &quot;3&quot; per mostrare la trama selezionata. </li><li> Fare clic sullo strumento &quot;Straight&quot; e tracciare le linee per separare l&#39;area segmentata. </li><li> Fare clic sullo strumento &quot;Bacchetta&quot; per registrare le misurazioni. </li><li> Esportare i dati. </li><li> Normalizzare i livelli di m 6 A con la 18S RNA ribosomiale banda dalla colorazione bromuro di 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>

Gli autori non hanno nulla da rivelare.

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 border="0" cellpadding="0" cellspacing="0" width="598">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNaseZap solution</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9782</td>
			<td style="width:167px;">Protocol 1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TRI Reagent solution</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9738</td>
			<td>Protocol 1.2.1.1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1-Bromo-3-chloropropane (BCP)</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">B9673</td>
			<td>Protocol 1.2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:96px;">JTbaker</td>
			<td style="width:119px;">8006</td>
			<td>Protocol 1.2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopropanol</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">I9516</td>
			<td>Protocol 1.2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GenElute mRNA Miniprep Kit</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">MRN70</td>
			<td>Protocol 1.2.2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycogen</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9510</td>
			<td>Protocol 1.2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium acetate</td>
			<td style="width:96px;">Fluka</td>
			<td style="width:119px;">71183</td>
			<td>Protocol 1.2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nuclease-free water</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9930</td>
			<td>Protocol 1.2.2.6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DEPC</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">D5758</td>
			<td>Protocol 2.1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:96px;">JT Baker</td>
			<td style="width:119px;">A426</td>
			<td>Protocol 2.1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MOPS</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">M1254</td>
			<td>Protocol 2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">37% formaldehyde Solution</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">F8775</td>
			<td>Protocol 2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA, Disodium Salt</td>
			<td style="width:96px;">JT Baker</td>
			<td style="width:119px;">8993</td>
			<td>Protocol 2.1.3 10X MOPS buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">formamide</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">F7503</td>
			<td>Protocol 2.2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNA Millennium Marker</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM7150</td>
			<td>Protocol 2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethidium Bromide</td>
			<td style="width:96px;">Amresco</td>
			<td style="width:119px;">X328</td>
			<td>Protocol 2.2.5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ficoll 400</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">17-0300-10</td>
			<td>Protocol 2.2.5 tracking dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromophenol blue</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">114391</td>
			<td>Protocol 2.2.5 tracking dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylene Cyanol</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">X4126</td>
			<td>Protocol 2.2.5 tracking dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:96px;">JT Baker</td>
			<td style="width:119px;">3624</td>
			<td>Protocol 2.4.2 20X SSC buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trisodium citrate</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">S1804</td>
			<td>Protocol 2.4.2 20X SSC buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">filter paper</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN6101M</td>
			<td>Protocol 2.4.4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GE Hybond-N+ membrane</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN303B</td>
			<td>Protocol 2.4.6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stratalinker UV Crosslinker 2400</td>
			<td style="width:96px;">Stratagene</td>
			<td style="width:119px;">400075</td>
			<td>Protocol 3.1.2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gel Catcher 1500</td>
			<td style="width:96px;">ANT Technology</td>
			<td style="width:119px;">Gel Catcher 1500</td>
			<td>Protocol 3.1.5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-m6A (N6-methyladenosine)</td>
			<td style="width:96px;">Synaptic Systems</td>
			<td style="width:119px;">202003</td>
			<td>Protocol 3.2.3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amersham ECL Anti-Rabbit IgG, HRP-linkd whole Ab</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA934</td>
			<td>Protocol 3.2.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ChemiDoc MP System</td>
			<td style="width:96px;">BIO-RAD</td>
			<td style="width:119px;">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.

Dopo 14 giorni nel normale fase circadiana luce-buio, i topi wild-type sono stati collocati in buio costante. Gli RNA dal fegato sono stati campionati ad intervalli di 4 ore e studiate con modificato blotting settentrionale. La metilazione di rRNA, mRNA, e piccoli RNA erano chiaramente rilevata (Figura 2). Il confronto tra differenti tempi circadiani (CT) può essere accuratamente calcolata con lo standard 18S rRNA. C&#39;era robusta oscillazione circadiana di m 6 livelli A in rRNA, mRNA, e piccoli RNA. Per evitare l&#39;interferenza offerto da rRNA, RNA poliadenilato possono essere purificate, come al punto 1.2.2. Dopo la purificazione, l&#39;rRNA può essere sostanzialmente eliminato per consentire una migliore visualizzazione di altri RNA (Figura 3). <img alt="Figura 1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html> g&gt; Figura 1: Montaggio della unità di trasferimento macchia. è indicata l&#39;unità di trasferimento capillare per asciugare l&#39;RNA alla membrana. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/54672/54672fig2.jpg" /> Figura 2: il ritmo circadiano delle m 6 levels in C57BL / 6J. Questa figura mostra la m 6 A blot di RNA totale da fegati di topi wild-type sacrificato il primo giorno della fase dark-scuro dopo 14 giorni di una normale fase leggera-buio a 4 h intervalli. La quantificazione è stata effettuata utilizzando il m 6 A differenza di densità delle immagini tra 18S e 28S rRNA. La m 6 A abbondanza stato normalizzato alla banda 18S rRNA dal gel formaldeide in basso.et = &quot;_ blank&quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="Figura 3" src="/files/ftp_upload/54672/54672fig3.jpg" /> Figura 3: I m 6 una macchie con o senza rRNA. Rappresentante m sono mostrati 6 una macchie del totale RNA e l&#39;RNA poliadenilato dal fegato di topi wild-type. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa 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 tampone MOPS (pH 7,0, proteggere dalla luce) </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"> Acetato di sodio </td><td> 4.1 </td><td> g </td><td>0,05 M </td></tr><tr><td></td><td colspan="3"> EDTA, Disodium Salt </td><td> 3.7 </td><td> g </td><td> 0,01 M </td></tr><tr><td></td><td colspan="3"> acqua DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Totale </td><td> 1 </td><td> L </td><td></td></tr><tr><td></td><td colspan="3"> Mescolare a temperatura ambiente </td><td></td><td></td><td></td></tr><tr><td> 2 </td><td colspan="3"> 20x tampone SSC (pH 7,0) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Cloruro di sodio </td><td> 175,3 </td><td> g </td><td> 3 M </td></tr><tr><td></td><td colspan="3"> citrato trisodico </td><td> 88.3 </td><td> g </td><td> 0.3 M </td></tr><tr><td></td><td colspan="3"> acqua DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Totale </td><td> 1 </td><td> L </td><td></td&gt; </tr><tr><td></td><td colspan="3"> Mescolare a temperatura ambiente, poi in autoclave </td><td></td><td></td><td></td></tr><tr><td> 3 </td><td colspan="3"> Dye monitoraggio </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Buffer 10x MOPS </td><td> 500 </td><td> microlitri </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"> bromofenolo blu </td><td> 0.01 </td><td> g </td><td> 0,2% </td></tr><tr><td></td><td colspan="3"> xilene cianolo </td><td> 0.01 </td><td> g </td><td> 0,2% </td></tr><tr><td></td><td colspan="3"> acqua DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Totale </td><td> 5 </td><td> mL </td><td></td></tr><tr><td></td><td colspan="3"> Conservare a -20 ° C </td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> acqua DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Diluire il rapporto a 1: 1.000 di DEPC in DDH 2 O </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Mescolare notte a temperatura ambiente, poi in autoclave </td><td></td><td></td><td></td></tr><tr><td> 5 </td><td colspan="3"> tampone campione (riparo dalla luce) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Buffer 10x MOPS </td><td> 200 </td><td> microlitri </td><td></td></tr><tr><td></td><td colspan="3"> 37% di formaldeide </td><td> 270 </td><td> microlitri </td><td></td></tr><tr><td></td><td colspan="3"> formammide </td><td> 660 </td><td> microlitri </td><td></td></tr><tr><td></td><td colspan="3"> Conservare a -20 ° C </td><td></td><td></td><td></td></tr></tbody&gt; </table> Tabella 1: Tamponi e soluzioni.

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

Un metodo di blotting nord modificato per misurare N 6 -methyladenosine (m 6 a) delle modifiche in RNA è descritto. L&#39;attuale metodo in grado di rilevare le modifiche in diversi RNA e controlli sotto vari disegni sperimentali.

Un metodo per l&#39;RNA di misura<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine modifiche di cellule e tessuti

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

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Nanyang Technological University

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Biochemistry

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

10.8K Views.  Chang Gung Memorial Hospital, Chang Gung University College of Medicine.  Un metodo di blotting nord modificato per misurare N 6 -methyladenosine (m 6 a) delle modifiche in RNA è descritto. L'attuale metodo in grado di rilevare le modifiche in diversi RNA e controlli sotto vari disegni sperimentali. 

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

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

Un metodo efficace per l&#39;isolamento di RNA altamente purificato da semi per l&#39;utilizzo in Quantitative Analysis trascrittoma

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

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Biochimica

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Un metodo per misurare il metabolismo in Ordinati sottopopolazioni di cellule Comunità complessi utilizzando Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Isolamento di proteine ​​filamentose intermedie da tessuti multipli di topo per studiare modificazioni post-traduzionali associate all&#39;invecchiamento

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Preparazione dei campioni per l'identificazione basati sulla spettrometria di massa di RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Un metodo colorimetrico per la misurazione del ferro contenuto nelle piante

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Toeprinting analisi di traduzione Iniziazione formazione complessa su mRNAs mammiferi

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

Microgravimetry dissipativo per studiare la dinamica di associazione del fosfolipide proteina Annessina A2 a lipidici solido-supportato utilizzando un risonatore al quarzo

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Misurare e interpretare i tassi di consumo di ossigeno in tutto Vola testa segmenti

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

Visualizzazione fluorescente dell'RNA marcato al mango nei gel di poliacrilofofora tramite un metodo poststaino

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

Quantificazione del Coenzevia A nelle cellule e nei tessuti

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...