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>

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

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

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

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="http://ecsource.jove.com/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="http://ecsource.jove.com/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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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 In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

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

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

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

Un test In Vitro per rilevare attività di tRNA-Isopentenyl Transferase

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

Ricerca

Biochimica

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

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

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

Analisi anticorp-free per l'analisi dell'attività di RNA methyltransferase

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

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

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

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

Migliorare il piccolo RNA-seq: meno Bias e migliore rilevamento di 2'-O-Methyl RNA

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

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

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

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

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

Test di immunoprecipitazione dell'RNA metilato per studiare m5C Modifica in Arabidopsis

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

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

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

<meta charset="utf8"></head><body>2D-HELS MS Seq: Un metodo generale basato su LC-MS per il sequenziamento diretto e de novo di miscele di RNA con diverse modifiche nucleotidiche

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

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

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

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

Esplorazione del metiloma dell'arginina mediante spettroscopia di risonanza magnetica nucleare

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

Ricerca sul cancro

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

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

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

Preparazione del campione dell'impianto per la misurazione del contenuto nucleoside/nucleotidico con hplc-MS/MS

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

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

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

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

Analisi dipendente da DNAzyme del rRNA 2'-O-Metilazione

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

Biologia dello sviluppo

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

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

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

Caratterizzazione delle modifiche dell'RNA in singoli neuroni utilizzando la spettrometria di massa

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

Un metodo per l'RNA di misura

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Test di immunoprecipitazione dell'RNA metilato per studiare m⁵C Modifica in Arabidopsis

2D-HELS MS Seq: Un metodo generale basato su LC-MS per il sequenziamento diretto e de novo di miscele di RNA con diverse modifiche nucleotidiche

2D-HELS MS Seq: Un metodo generale basato su LC-MS per il sequenziamento diretto e de novo di miscele di RNA con diverse modifiche nucleotidiche

Esplorazione del metiloma dell'arginina mediante spettroscopia di risonanza magnetica nucleare

Preparazione del campione dell'impianto per la misurazione del contenuto nucleoside/nucleotidico con hplc-MS/MS

Analisi dipendente da DNAzyme del rRNA 2'-O-Metilazione

Caratterizzazione delle modifiche dell'RNA in singoli neuroni utilizzando la spettrometria di massa