Name: a method for measuring rna n6-methyladenosine modifications in cells and tissues
Uploaded: 2016-12-05T13:00:00
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Description: Watch this Scientific Journal Video about A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues at JoVE.com

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).

MERK: total RNA m 6 A nivå inkluderer rRNA, mRNA, og andre små RNA. Siden ribosomalt RNA har rikelig m 6 A modifikasjoner, måling av m 6 A nivåer må vurdere dette faktum. 1. RNA Isolation <ol><li> Bruke ribonuklease dekontaminering løsning (sprayet på tørkepapir), tørk av pipetter og lab benk overflater. Våt papirhåndklær med nukleasefritt vann og tørk av pipetter og lab benk overflater igjen. </li><li> RNA Utvinning <ol><li> Total RNA Isolation <ol><li> Ved hjelp av en overliggende rører, homogen 50-100 mg vev eller 5-10 x 10 6 celler i 1 ml av RNA isolering oppløsning holdt ved 4 ° C. MERK: Mer vev (100-150 mg) kan være nødvendig for adipose vevsprøver fra mus på grunn av de lavere RNA-konsentrasjoner i denne. Prøver hentet fra mus hjerte, lever, skjelettmuskulatur, lunge, hjerne, og makrofager har vært ansatt tidligere fire. </li><li> Icubate eksempel homogenater i 5 min ved romtemperatur. </li><li> Tilsett 100 ul av 1-brom-3-klorpropan (BCP) per 1 mL prøve homogenat. Rist rørene kraftig i 15 sekunder og videre for å isolere total RNA i henhold til produsentens instruksjoner 24. </li></ol></li><li> Polyadenylert mRNA Rensing fra Isolert Total RNA <ol><li> Rens polyadenylerte mRNA ved hjelp av tilgjengelige sett eller protokoller. <ol><li> Rist godt for å re-suspendere hver av følgende løsninger: 2x bindende løsning, oligo (dT) isopor perler, og vaskeløsning. Kontroller at oligo (dT) perler varmes opp til romtemperatur før bruk. </li><li> Overfør 120 ul av elueringsoppløsningen pr preparat til et rør og oppvarm til 70 ° C i en varmeblokk. </li><li> Pipettere opp til 500 mikrogram av total RNA inn i et mikrosentrifugerør. Med nukleasefritt vann, justere volumet til 250 mL. </li><li> Tilsett 250 ul av 2x binding løsning av total RNA såning og vortex kort for å blande innholdet. </li><li> Tilsett 15 mL av oligo (dT) perler og virvle grundig å blande. </li><li> Inkuber blandingen ved 70 ° C i 3 minutter. </li><li> Fjern prøven fra varmeblokken og la det stå ved romtemperatur i 10 min. </li><li> Sentrifuger ved 15000 x g i 2 min. </li><li> Fjern forsiktig supernatanten, etterlot ca 50 mL. </li><li> Legg 500 mL vaskeløsning for å re-suspendere pellet ved pipettering. </li><li> Pipetter suspensjonen i en spin filter / samling rør sett. </li><li> Sentrifuger ved 15000 x g i 2 min. Fjerne kolonnen fra oppsamlingsrøret og kast gjennomstrømning. Returnere kolonnen til oppsamlingsrøret. </li><li> Pipetter 500 mL av vaskeløsning på spin-filter. </li><li> Sentrifuger ved 15000 x g i 2 min. </li><li> Overfør spinnfilter inn i en ny samling rør. </li><li> Pipetter 50 ul av elueringsoppløsningen oppvarmettil 70 ° C på midten av spinn filteret. </li><li> Inkuber i 2-5 minutter ved 70 ° C. Sentrifuger ved 15000 x g i 1 min. </li><li> Pipetter en ytterligere 50 ul av elueringsoppløsningen oppvarmet til 70 ° C på midten av spinn filteret. </li><li> Gjenta trinn 1.2.2.1.18. </li></ol></li><li> Tilsett 100 mg / ml glykogen, 0,1 volum 3 M natriumacetat buffer (pH 5,2) og 2,5 volumer absolutt etanol. Utfelles over natten ved -80 ° C. </li><li> Sentrifuger ved 15000 x g i 25 min ved 4 ° C. Kast supernatanten. </li><li> Vask pelleten med 1 ml 75% etanol. Sentrifuger ved 15000 x g i 15 min ved 4 ° C. Fjern forsiktig etanol. </li><li> Tørr i luft i 3-5 min. </li><li> Tilsett 10 ul av nuklease-fritt vann for å oppløse pelleten. </li><li> Oppbevar ved -70 ° C. </li></ol></li></ol></li><li> RNA Kvalifisering og Kvantifisering <ol><li> Ved hjelp av et spektrofotometer, bestemme RNA-konsentrasjonen med NotIng av absorbans ved 260 nm og 280 nm. Forholdet A 260 / A 280 bør være 1,8-2,2. </li><li> Sjekk RNA prøven kvalitet ved hjelp av 0,8% agarose gelelektroforese 25. MERK: intakt eukaryote total RNA bør vise intense 28S og 18S rRNA band. Forholdet mellom 28S versus 18S rRNA-band intensitet for en god RNA forberedelse er ~ 2. </li></ol></li></ol> 2. Gel elektroforese og Transfer MERK: buffer forberedelse protokoller er gitt i tabell 1. <ol><li> Formaldehyd Gel Forberedelse (1%) <ol><li> Skyll alle elektroforese utstyr med dietylpyrokarbonat (DEPC) vann. </li><li> Smelt 2,5 g agarose i 215 ml DEPC vann helt i en mikrobølgeovn. </li><li> Legg 12,5 ml 10x 3- (N- morfolino) propansulfonsyre (MOPS) buffer og 22,5 ml 37% formaldehyd. </li><li> Helle den inn i elektroforese apparat med en 1,5 mm tykk kam. </li><li> Eliminer bobler eller push them til kantene av gelen med en ren kam. </li><li> La gelen stivne ved romtemperatur. </li></ol></li><li> Prøvepreparering <ol><li> Bland 1-10 ug av total RNA og 11,3 ul prøvebuffer. </li><li> Bland 2 ug av RNA-markør (1 ug / ul) med 11,3 mL av prøvebuffer. </li><li> Legg nuklease-fritt vann til prøvene fra 2.2.1 og 2.2.2 til et totalt volum på 16 ul. </li><li> Oppvarm til 60 ° C i 5 minutter, og deretter koble på is. </li><li> Bland 16 ul av prøven fra 2.2.3 med 4 pl av sporende farvestoff, som inneholder 0,1 ug / ul etidiumbromid på is. FORSIKTIG: Etidiumbromid er muligens teratogen og giftig ved innånding. Les etidiumbromid sikkerhetsdatabladet. </li></ol></li><li> elektroforese <ol><li> Tilsett 200 ml 10x MOPS buffer til 1800 ml DDH 2 O for å lage en 1x MOPS buffer. </li><li> Bruk 1x MOPS buffer for å skylle brønnene i gelen. </li><li> Pre-run than gel ved 20 V i 5 minutter. </li><li> Laste RNA prøver til brønnene i gelen. </li><li> Dekk med folie for å unngå lyseksponering. Kjør på 35 V i ca 17 timer (over natten) </li><li> Undersøke og fotografere gelen under UV-lys. </li></ol></li><li> Overføre <ol><li> Kutt gel for å fjerne den ubrukte delen. </li><li> Fremstille 500 ml av 10x SSC-buffer (250 ml 20x SSC-buffer og 250 ml DEPC vann). </li><li> Vask gelen to ganger med 10 x SSC-buffer i 20 minutter med risting ved 50 rpm. </li><li> Kuttes en filterpapirarket stor nok til å tjene som en veke papir, som absorberer overføringsbuffer fra overføringsmagasinet (figur 1). </li><li> Skjær 4 stykker av filterpapir til samme størrelse som gel. </li><li> Skjær en plate av positivt ladet nylonmembran til samme størrelse som den gel. </li><li> Marker et hakk på gelen og membranen som en markør for å sikre riktig retning. </li><li> Sug membranen i 2x SSC buffer i 15 min. </li><li> Hell 500 ml of 10x SSC buffer i overføringen skuffen. </li><li> Lå den store filterpapirarket på tvers av glassplaten og våt filterpapiret med overføringsbuffer. </li><li> Rull ut noen bobler med en pipette. </li><li> Sug 2 stykker av pre-cut filter papir i overføringsbuffer og plassere dem på midten av filterpapiret fra trinn 2.4.5. Fjern eventuelle bobler. </li><li> Legg gelen opp-ned på filterpapiret. </li><li> Lå membranen på toppen av gelen. Juster hakkene av gelen og membranen. </li><li> Legg 2 stykker av pre-cut filter papir på toppen av membran og fukte filterpapiret med overføringsbuffer. </li><li> Fjern eventuelle bobler mellom lagene. </li><li> Påfør plastfolie rundt gel for å sikre at håndklær bare suge opp buffer som passerer gjennom gelen og membranen. </li><li> Stable papirhåndklær på toppen av filterpapiret. </li><li> Plasser en overdimensjonert glassplate på toppen av håndklær. </li><li> Plasser en vekt på toppen av stabelen. </li><li>Hold over natten for å tillate den å overføre RNA fra gelen til membranen. </li></ol></li></ol> 3. Påvisning av N 6 -methyladenosine <ol><li> membran Kryssbinding <ol><li> Hold membranen fuktig i 2x SSC buffer. </li><li> Legg 2 ark med filter papir nedsenket med 10x SSC buffer i UV tverrbinder. </li><li> Plasser membran på toppen av filterpapiret med RNA-adsorberte siden vendt opp. </li><li> Velg &quot;autocrosslink mode&quot; (120.000 μJ) og trykk på &quot;Start&quot; -knappen for å starte bestråling. </li><li> Undersøke membranen og gelen med et UV-gel bildefangeren for å bekrefte at RNA-overføring fra gelen til membranen. </li></ol></li><li> immunoblotting <ol><li> Blokkere membranen i 5% ikke-fettholdig melk i Tris-bufret saltvann med Tween 20 (TBST) buffer ved romtemperatur i 1 time. </li><li> Vask tre ganger med TBST-buffer i 15 min. </li><li> Inkuber membran over natten i m 6 A (&lt;em&gt; N 6 -methyladenosine) antistoffoppløsning (1: 1000 i 5% ikke-fettholdig melk TBST-buffer) ved 4 ° C. </li><li> Vask membranen tre ganger med TBST-buffer i 15 min. </li><li> Inkuber membranen i det HRP-konjugert esel-anti-kanin-antistoff-løsning (1: 2000 i 5% ikke-fettholdig melk TBST-buffer) i 1 time ved romtemperatur. </li><li> Vask membranen tre ganger med TBST-buffer i 15 min. </li><li> Påfør forbedret kjemiluminescenssubstrat (0,125 ml per cm 2 av membran). </li><li> Fang chemiluminescence med de optimale innstillingene for det digitale imager, i henhold til produsentens instruksjoner 26. </li></ol></li><li> m 6 A Kvantifisering <ol><li> Mål den relative m 6 A kjemiluminescens intensitet bruker ImageJ programvare. <ol><li> Under ImageJ programvare-menyen, velg &quot;File&quot; for å åpne den aktuelle filen. </li><li> Velg &quot;rektangel&quot; verktøyet fra ImageJ og tegn en ramme rundtsignalene. </li><li> Hvis ribosomalt RNA er ikke fokus for eksperimentene, unngå 18S og 28S ribosomalt RNA m 6 A band for beregningen. </li><li> Klikk &quot;Command&quot; og &quot;1&quot; for å bekrefte den valgte kjørefelt. </li><li> Trykk &quot;Command&quot; og &quot;3&quot; for å vise den valgte handlingen. </li><li> Klikk på &quot;Straight&quot; -verktøyet og trekke linjer til å skille det segmenterte området. </li><li> Klikk på &quot;Wand&quot; verktøy for å registrere målingene. </li><li> Eksportere data. </li><li> Normalisere nivået av m 6 A med den 18S ribosomale RNA-bånd fra etidiumbromidfarging. </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>

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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">
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		<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.

Etter 14 dager i vanlig lys-mørk circadian fase ble villtype-mus plasseres i konstant mørke. De RNA fra leveren ble samplet med 4 timers mellomrom, og studerte med modifisert nordblotting. Den metylering av rRNAs, mRNA, og små RNA ble tydelig påvist (figur 2). Sammenligningen mellom ulike cirkadianske ganger (CT) kan beregnes nøyaktig med 18S rRNA-standarden. Det var robust circadian svinging av m 6 A nivåer i rRNA, mRNA, og små RNA. For å unngå forstyrrelser bød av rRNA, kan polyadenylerte RNA bli renset, som i trinn 1.2.2. Etter rensing kan rRNA elimineres i stor grad for å tillate bedre visualisering av andre RNA (Figur 3). <img alt="Figur 1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html> g&gt; Figur 1: Montering av blot overføringsenheten. Den kapillære overføringsenhet for blotting av RNA til membranen er vist. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">Klikk her for å se en større versjon av dette tallet.</a> <img alt="Figur 2" src="/files/ftp_upload/54672/54672fig2.jpg" /> Figur 2: døgnrytme av m 6 A nivåer i C57BL / 6J mus. Denne figuren viser m 6 En blot av total RNA fra lever fra villtype mus avlivet på den første dagen av den mørke-mørke fasen etter 14 dager med en normal lys-mørke-fase ved 4 timers intervaller. Kvantifiseringen ble gjort ved hjelp av m 6 A bildetetthet forskjell mellom 18S og 28S rRNA. M 6 En overflod ble normalisert til 18S rRNA båndet fra formaldehyd gelen i bunnen.et = &quot;_ blank&quot;&gt; Klikk her for å se en større versjon av dette tallet. <img alt="Figur 3" src="/files/ftp_upload/54672/54672fig3.jpg" /> Figur 3: De m 6 A blotter med eller uten rRNA. Representative m 6 A-blot av total RNA og polyadenylert RNA fra leveren hos villtype-mus er vist. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">Klikk her for å se en større versjon av dette tallet.</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 buffer (pH 7,0, beskytte mot lys) </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, dinatriumsalt </td><td> 3.7 </td><td> g </td><td> 0,01 M </td></tr><tr><td></td><td colspan="3"> DEPC vann </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Total </td><td> 1 </td><td> L </td><td></td></tr><tr><td></td><td colspan="3"> Omrør ved romtemperatur </td><td></td><td></td><td></td></tr><tr><td> 2 </td><td colspan="3"> 20x SSC-buffer (pH 7,0) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Natriumklorid </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 vann </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Total </td><td> 1 </td><td> L </td><td></td&gt; </tr><tr><td></td><td colspan="3"> Omrør ved romtemperatur, deretter autoklav </td><td></td><td></td><td></td></tr><tr><td> 3 </td><td colspan="3"> sporing Dye </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10x MOPS buffer </td><td> 500 </td><td> mL </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"> bromfenolblå </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 vann </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Total </td><td> 5 </td><td> ml </td><td></td></tr><tr><td></td><td colspan="3"> Oppbevares ved -20 ° C </td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> DEPC vann </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Fortynn forhold på 1: 1000 av DEPC i DDH 2 O </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Omrør over natten ved romtemperatur, deretter autoklav </td><td></td><td></td><td></td></tr><tr><td> 5 </td><td colspan="3"> Sample buffer (beskytte mot lys) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10x MOPS buffer </td><td> 200 </td><td> mL </td><td></td></tr><tr><td></td><td colspan="3"> 37% formaldehyd </td><td> 270 </td><td> mL </td><td></td></tr><tr><td></td><td colspan="3"> formamide </td><td> 660 </td><td> mL </td><td></td></tr><tr><td></td><td colspan="3"> Oppbevares ved -20 ° C </td><td></td><td></td><td></td></tr></tbody&gt; </table> Tabell 1: Buffere og løsninger.

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

En modifisert northern-blotting fremgangsmåte for å måle N 6 -methyladenosine (m 6 A) modifikasjoner i RNA er beskrevet. Den nåværende metoden kan påvise endringer i ulike RNA og kontroller under ulike eksperimentelle design.

En metode for måling av RNA<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine Endringer i celler og vev

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

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