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).

NOTA: O total RNA m 6 A nível inclui rRNA, mRNA, e outros pequenos RNAs. Desde RNA ribossomal tem abundantes m 6 modificações A, a medição da m 6 levels terá de considerar este fato. Isolamento 1. RNA <ol><li> Usando a solução ribonuclease descontaminação (pulverizado sobre toalhas de papel), limpe as pipetas e superfícies das bancadas do laboratório. papel-toalha molhada com água livre de nuclease e limpar as pipetas e superfícies das bancadas do laboratório novamente. </li><li> Extração de RNA <ol><li> Isolamento de ARN total <ol><li> Usando um agitador suspenso, homogeneizar 50-100 mg de tecido ou 5-10 X 10 6 células em 1 mL de solução de isolamento de ARN mantidas a 4 ° C. NOTA: Mais tecido (100-150 mg) pode ser necessária para amostras de tecido adiposo de ratos por causa das concentrações de ARN inferiores das mesmas. As amostras provenientes de rato coração, fígado, músculo esquelético, o pulmão, cérebro, e os macrófagos foram empregues quatro anteriormente. </li><li> DentroCubate os homogenatos de amostra durante 5 minutos a temperatura ambiente. </li><li> Adicionar 100 uL de 1-bromo-3-cloropropano (BCP) por 1 ml de homogenato da amostra. Agitar vigorosamente os tubos durante 15 s e proceder para isolar o ARN total de acordo com as instruções do fabricante 24. </li></ol></li><li> A purificação a partir de ARNm poliadenilado do RNA total isolado <ol><li> Purificar mRNA poliadenilados usando kits ou protocolos disponíveis. <ol><li> Agitar minuciosamente para re-suspender cada uma das seguintes soluções: A solução de ligação, oligo (dT) de grânulos de poliestireno, e a solução de lavagem 2x. Certifique-se de que o oligo (dT) grânulos quentes à temperatura ambiente antes do uso. </li><li> Transferir 120 uL da solução de eluição por preparação para um tubo de calor e a 70 ° C num bloco de aquecimento. </li><li> Pipeta-se 500 ^ g de ARN total para um tubo de microcentrífuga. Com água livre de nuclease, ajustar o volume para 250 mL. </li><li> Adicionar 250 uL de solução de ligação 2x para o ARN total assimlução e agitar com vortex brevemente para misturar o conteúdo. </li><li> Adicionar 15 ul de oligo (dT) e grânulos de vórtice para misturar completamente. </li><li> Incubar a mistura a 70 ° C durante 3 min. </li><li> Retirar a amostra do bloco de aquecimento e deixou-se em repouso à temperatura ambiente durante 10 min. </li><li> Centrifugar a 15000 xg durante 2 min. </li><li> Remova cuidadosamente o sobrenadante, deixando para trás cerca de 50 mL. </li><li> Adicionar 500 uL de solução de lavagem para re-suspender o sedimento por pipetagem. </li><li> Pipetar a suspensão em um conjunto tubo de filtro de spin / coleção. </li><li> Centrifugar a 15000 xg durante 2 min. Retirar a coluna do tubo de recolha e o descarte de fluxo. Retornar a coluna para o tubo de recolha. </li><li> Pipetar 500 L de solução de lavagem para o filtro de rotação. </li><li> Centrifugar a 15.000 x g durante 2 min. </li><li> Transferência do filtro de rotação para um tubo de recolha de fresco. </li><li> Pipetar 50 ul de solução de eluição aquecidaa 70 ° C para o centro do filtro de rotação. </li><li> Incubar durante 2-5 min a 70 ° C. Centrifugar a 15000 xg durante 1 min. </li><li> Pipetar um adicional de 50 ul de solução de eluição aquecida a 70 ° C para o centro do filtro de rotação. </li><li> Repita o passo 1.2.2.1.18. </li></ol></li><li> Adicionam-se 100 ug / mL de glicogénio, 0,1 volume de tampão de acetato de sódio 3 M (pH 5,2), e 2,5 volumes de etanol absoluto. Precipitar durante a noite a -80 ° C. </li><li> Centrifuga-se a 15000 xg durante 25 min a 4 ° C. Descartar o sobrenadante. </li><li> Lava-se a pelete com 1 ml de etanol a 75%. Centrifuga-se a 15000 xg durante 15 min a 4 ° C. Retire cuidadosamente o etanol. </li><li> Seco ao ar durante 3-5 min. </li><li> Adicionar 10 ul de água isenta de nuclease para dissolver o sedimento. </li><li> Armazenar a -70 ° C. </li></ol></li></ol></li><li> RNA qualificação e quantificação <ol><li> Usando um espectrofotómetro para determinar a concentração de ARN por NotIng a absorvância a 260 nm e 280 nm. A relação A260 / A280 deve ser 1,8-2,2. </li><li> Verifique a qualidade da amostra de RNA usando 0,8% eletroforese em gel de agarose 25. NOTA: O RNA total eucariótica intacta deve mostrar intensas bandas de 28S e 18S rRNA. A proporção de 28S contra 18S rRNA intensidade da banda para uma boa preparação de RNA é ~ 2. </li></ol></li></ol> 2. eletroforese em gel e Transferência NOTA: Os protocolos de preparação tampão são dadas na Tabela 1. <ol><li> Formaldeído Preparação de gel (1%) <ol><li> Lavar todo o equipamento de eletroforese com dietilpirocarbonato (DEPC) de água. </li><li> Melt 2,5 g de agarose em 215 ml de água com DEPC completamente num forno de microondas. </li><li> Adicionar 12,5 ml de 10x 3- (N- morfolino) propanossulfónico (MOPS) Tampão e 22,5 ml de formaldeído a 37%. </li><li> Deite-o no aparelho de electroforese com um pente de espessura de 1,5 mm. </li><li> Eliminar as bolhas ou empurrar tbainha para as bordas do gel com um pente limpo. </li><li> Deixar o gel solidificar à temperatura ambiente. </li></ol></li><li> Preparação de amostra <ol><li> Misture 1-10 ug de ARN total, e 11,3 ul de tampão de amostra. </li><li> Misturar 2 ug do marcador de ARN (1 mg / mL) com 11,3 mL de tampão de amostra. </li><li> Adicionar água isenta de nuclease para as amostras a partir de 2.2.1 e 2.2.2 para um volume total de 16 uL. </li><li> Aquecer a 60 ° C durante 5 min, e em seguida esfriar em gelo. </li><li> Misturar 16 ul da amostra a partir de 2.2.3 com 4 ul de corante de acompanhamento, que contém 0,1 mg / mL de brometo de etidio em gelo. CUIDADO: O brometo de etídio é, possivelmente, teratogênico e tóxico se inalado. Leia brometo de etídio segurança folha de dados. </li></ol></li><li> eletroforese <ol><li> Adicione 200 ml de 10x tampão MOPS a 1.800 mL de DDH 2 O para fazer um tampão de corrida 1x MOPS. </li><li> Utilize tampão de corrida 1x MOPS para lavar os poços do gel. </li><li> Pré-run tele gel a 20 V durante 5 minutos. </li><li> Coloque as amostras de ARN para os poços do gel. </li><li> Cubra com papel alumínio para evitar a exposição à luz. Execute a 35 V durante cerca de 17 h (durante a noite) </li><li> Examinar e fotografar o gel sob uma luz UV. </li></ol></li><li> Transferir <ol><li> Cortar o gel para remover a porção não utilizada. </li><li> Preparar 500 ml de 10x tampão SSC (250 ml de 20x tampão SSC e 250 mL de água com DEPC). </li><li> Lavar o gel duas vezes com 10x tampão SSC durante 20 min com agitação a 50 rpm. </li><li> Cortar uma folha de papel de filtro suficientemente grande para servir como um pavio de papel, que absorve tampão de transferência do tabuleiro de transferência (figura 1). </li><li> Corte 4 pedaços de papel de filtro para o mesmo tamanho que o gel. </li><li> Cortar uma folha de membrana de nylon carregada positivamente para o mesmo tamanho que o gel. </li><li> Marcar um entalhe sobre o gel e a membrana, como um marcador para assegurar a orientação correcta. </li><li> Embeber a membrana em tampão SSC 2x durante 15 min. </li><li> Despeje 500 ml of 10x SSC tampão na bandeja de transferência. </li><li> Coloque a folha de papel de filtro grande do outro lado da placa de vidro e molhar o papel de filtro com tampão de transferência. </li><li> Rolar para fora todas as bolhas com uma pipeta. </li><li> Soak 2 peças de papel de filtro pré-corte em tampão de transferência e coloca-os no centro do papel de filtro a partir do passo 2.4.5. Remover quaisquer bolhas. </li><li> Lay topo do lado do gel para baixo no papel de filtro. </li><li> Colocar a membrana no topo do gel. Alinhar os entalhes do gel e da membrana. </li><li> Coloque 2 peças de papel de filtro pré-corte na parte superior da membrana e molha o filtro de papel com tampão de transferência. </li><li> Remover quaisquer bolhas entre as camadas. </li><li> Aplicar película de plástico em torno do gel para garantir que as toalhas apenas absorver tampão que passa através do gel e da membrana. </li><li> Empilhar as toalhas de papel no topo do papel de filtro. </li><li> Coloque uma placa de vidro de grandes dimensões em cima das toalhas. </li><li> Coloque um peso no topo da pilha. </li><li>Manter durante a noite para permitir que o ARN de transferência do gel para a membrana. </li></ol></li></ol> 3. Detecção de N 6 -methyladenosine <ol><li> membrana de reticulação <ol><li> Mantenha a membrana úmida em tampão 2x SSC. </li><li> Coloque 2 folhas de papel de filtro imerso com 10x tampão SSC em reticulador de UV. </li><li> Colocar a membrana no topo do papel de filtro, com o lado da ARN-adsorvido voltado para cima. </li><li> Selecione &quot;Modo autocrosslink&quot; (120.000 μJ) e pressione o botão &quot;Iniciar&quot; para iniciar a irradiação. </li><li> Examinar a membrana e o gel com uma imagem do gel de UV coletor para confirmar a transferência de ARN do gel para a membrana. </li></ol></li><li> immunoblotting <ol><li> Bloquear a membrana em leite sem gordura a 5% em solução salina tamponada com Tris com Tween 20 (TBST) de tampão à temperatura ambiente durante 1 h. </li><li> Lavar três vezes com tampão de TBST, durante 15 min. </li><li> Incubar a membrana durante a noite em 6 m A (&lt;em&gt; solução de N 6 -methyladenosine) anticorpo (1: não gordo leite tampão TBST 1000 em 5%) a 4 ° C. </li><li> Lava-se a membrana três vezes com tampão de TBST, durante 15 min. </li><li> Incubar a membrana na solução de burro conjugado com HRP anti-anticorpo de coelho (1: 2000 em tampão TBST leite 5% sem gordura) durante 1 h à temperatura ambiente. </li><li> Lava-se a membrana três vezes com tampão de TBST, durante 15 min. </li><li> Aplicar o substrato quimioluminescente melhorado (0,125 ml por cm2 de membrana). </li><li> Capture a quimiluminescência com as definições ideais de o gerador de imagens digitais, de acordo com as instruções do fabricante 26. </li></ol></li><li> m 6 A Quantificação <ol><li> Medir a 6 m Uma intensidade quimioluminescente relativa utilizando o software ImageJ. <ol><li> Sob o menu do software ImageJ, selecione a opção &quot;File&quot; para abrir o arquivo pertinente. </li><li> Selecione a ferramenta &quot;Retângulo&quot; de ImageJ e desenhar uma moldura em voltaos sinais. </li><li> Se RNA ribossomal não é o foco dos experimentos, evitar os 18S e 28S RNA ribossomal m 6 a bandas para o cálculo. </li><li> Clique &quot;Comando&quot; e &quot;1&quot; para confirmar a pista seleccionada. </li><li> Pressione &quot;Command&quot; e &quot;3&quot; para mostrar o enredo selecionado. </li><li> Clique no botão &quot;Straight&quot; ferramenta e desenhe linhas para separar a área segmentada. </li><li> Clique na ferramenta &quot;Wand&quot; para gravar as medições. </li><li> Exportar os dados. </li><li> Normalizar os níveis de M 6 um com o ARN ribossómico 18S banda da coloração com brometo de etídio. </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>

Os autores não têm nada para revelar.

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

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

<table 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.

Após 14 dias em fase circadiana luz-escuro normais, os ratinhos do tipo selvagem foram colocadas em escuridão constante. Os ARN de fígado foram amostrados a intervalos de 4 horas e estudaram com Northern blotting modificado. A metilação de ARNr, ARNm, e pequenos RNAs foram claramente detectadas (Figura 2). A comparação entre os diferentes tempos circadianos (CT) pode ser calculado com precisão com o padrão de rRNA 18S. Houve oscilação circadiana robusto de m 6 levels em rRNA, mRNA, e pequenos RNAs. Para evitar a interferência oferecida por ARNr, RNAs poliadenilados pode ser purificado, tal como no passo 1.2.2. Após purificação, o rRNA pode ser largamente eliminada para permitir uma melhor visualização de outros ARN (Figura 3). <img alt="figura 1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html> g&gt; Figura 1: Montagem da unidade de transferência blot. A unidade de transferência capilar para blotting do ARN para a membrana é mostrado. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/54672/54672fig2.jpg" /> Figura 2: Ritmo circadiano dos m 6 levels em camundongos C57BL / 6J. Esta figura mostra a M 6 Um blot de ARN total a partir de fígados de ratinhos de tipo selvagem sacrificados no primeiro dia da fase de escuro-escuro após 14 dias de uma fase de luz-escuro normal em intervalos de 4 h. A quantificação foi feita usando o m 6 A diferença de densidade de imagem entre 18S e 28S rRNA. O M 6 Uma abundância foi normalizada para a banda de ARNr 18S a partir do gel de formaldeído na parte inferior.et = &quot;_ blank&quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 3" src="/files/ftp_upload/54672/54672fig3.jpg" /> Figura 3: Os m 6 borrões com ou sem rRNA. M representativas 6-blots de ARN total e ARN poliadenilado a partir de fígados de ratinhos do tipo selvagem são mostradas. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta 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"> tampão 10x MOPS (pH 7,0, proteger da luz) </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"> O acetato de sódio </td><td> 4.1 </td><td> g </td><td>0,05 M </td></tr><tr><td></td><td colspan="3"> EDTA, sal dissódico </td><td> 3.7 </td><td> g </td><td> 0,01 M </td></tr><tr><td></td><td colspan="3"> água DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Total </td><td> 1 </td><td> eu </td><td></td></tr><tr><td></td><td colspan="3"> Agita-se à temperatura ambiente </td><td></td><td></td><td></td></tr><tr><td> 2 </td><td colspan="3"> tampão SSC 20x (pH 7,0) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> O cloreto de sódio </td><td> 175,3 </td><td> g </td><td> 3 M </td></tr><tr><td></td><td colspan="3"> citrato trissódico </td><td> 88,3 </td><td> g </td><td> 0,3 M </td></tr><tr><td></td><td colspan="3"> água DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Total </td><td> 1 </td><td> eu </td><td></td&gt; </tr><tr><td></td><td colspan="3"> Agita-se à temperatura ambiente, em seguida, autoclave </td><td></td><td></td><td></td></tr><tr><td> 3 </td><td colspan="3"> Acompanhando Dye </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> tampão 10x MOPS </td><td> 500 </td><td> ul </td><td> 1x </td></tr><tr><td></td><td colspan="3"> Ficoll 400 </td><td> 0,75 </td><td> g </td><td> 15% </td></tr><tr><td></td><td colspan="3"> azul de bromofenol </td><td> 0,01 </td><td> g </td><td> 0,2% </td></tr><tr><td></td><td colspan="3"> xileno cianol </td><td> 0,01 </td><td> g </td><td> 0,2% </td></tr><tr><td></td><td colspan="3"> água DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Total </td><td> 5 </td><td> mL </td><td></td></tr><tr><td></td><td colspan="3"> Armazenar a -20 ° C </td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> água DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Diluir proporção de 1: 1000 de DEPC em ddH2O </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Agita-se durante a noite à temperatura ambiente, em seguida, autoclave </td><td></td><td></td><td></td></tr><tr><td> 5 </td><td colspan="3"> tampão de amostra (proteger da luz) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> tampão 10x MOPS </td><td> 200 </td><td> ul </td><td></td></tr><tr><td></td><td colspan="3"> 37% de formaldeído </td><td> 270 </td><td> ul </td><td></td></tr><tr><td></td><td colspan="3"> formamida </td><td> 660 </td><td> ul </td><td></td></tr><tr><td></td><td colspan="3"> Armazenar a -20 ° C </td><td></td><td></td><td></td></tr></tbody&gt; </table> Tabela 1: Os tampões e soluções.

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

Um método de transferência Northern modificado para medir N 6 -methyladenosine (M 6) Um modificações em RNA é descrito. O actual método pode detectar modificações em diversos RNAs e controles sob vários modelos experimentais.

Um método para medir RNA<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine modificações nas células e tecidos

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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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

Microgravimetry dissipativo para estudar a dinâmica da vinculação do fosfolipídeo proteína anexina A2 para Bilayers do Lipid com suporte sólido usando um ressonador de quartzo

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

Medindo e interpretando a taxas de consumo de oxigênio em toda a cabeça segmentos de voar

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

Visualização fluorescente da Mango-Tagged RNA em géis de poliacrilamida através de um método de pós-coloração

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

Quantificação da coenzima A em células e tecidos

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