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).

BEMÆRK: total RNA m 6 A niveau omfatter rRNA, mRNA, og andre små RNA. Da ribosomale RNA har rigeligt m 6 A modifikationer, måling af m 6 A-niveauer bliver nødt til at overveje dette faktum. 1. RNA-isolering <ol><li> Brug af ribonuklease dekontaminering løsning (sprøjtes på papirservietter), tørre pipetter og lab bænk overflader. Våde papirservietter med nuklease-frit vand og tørre ned pipetter og lab bænk overflader igen. </li><li> RNA-ekstraktion <ol><li> Total RNA Isolation <ol><li> Ved anvendelse af en overliggende omrører, homogenisere 50-100 mg væv eller 5-10 x 10 6 celler i 1 ml RNA-isolering opløsning holdes ved 4 ° C. BEMÆRK: Mere væv (100-150 mg) kan være nødvendig for adipose vævsprøver fra mus på grund af de lavere RNA koncentrationer deri. Prøver stammer fra muse hjerte, lever, skeletmuskel, lunge, hjerne, og makrofager har tidligere været ansat 4. </li><li> Icubate prøven homogenater i 5 minutter ved stuetemperatur. </li><li> Tilsæt 100 pi af 1-brom-3-chlorpropan (BCP) pr 1 ml prøve homogenat. Ryst rørene kraftigt i 15 s og fortsæt til isolere total RNA ifølge producentens anvisninger 24. </li></ol></li><li> Polyadenyleret mRNA Oprensning fra isolerede Total RNA <ol><li> Rens polyadenylerede mRNA hjælp tilgængelige kits eller protokoller. <ol><li> Ryst grundigt for at re-suspendere hver af følgende løsninger: 2x bindende løsning, oligo (dT) polystyren perler, og vask løsning. Sørg for, at oligo (dT) perler opvarmes til stuetemperatur før brug. </li><li> Overfør 120 pi elueringsopløsning pr forberedelse til et rør og opvarmes til 70 ° C i en varmeblok. </li><li> Pipette op til 500 ug totalt RNA i et mikrocentrifugerør. Med nuklease-frit vand, justere lydstyrken til 250 uL. </li><li> Tilsættes 250 pi af 2x binding løsning på det totale RNA sålution og kortvarigt vortex at blande indholdet. </li><li> Tilføj 15 uL af oligo (dT) perler og vortex grundigt at blande. </li><li> Inkuber blandingen ved 70 ° C i 3 minutter. </li><li> Fjern prøven fra varmeenheden og lad det stå ved stuetemperatur i 10 min. </li><li> Centrifuger ved 15.000 xg i 2 min. </li><li> Fjern forsigtigt supernatanten, efterlod ca. 50 uL. </li><li> Tilføj 500 pi vaskeopløsning at re-suspendere pellet ved pipettering. </li><li> Pipettere suspensionen i et spin filter / opsamlingsrør sæt. </li><li> Centrifuger ved 15.000 xg i 2 min. Fjern kolonne fra opsamlingsrøret og kassér gennemløbet. Retur kolonnen til opsamlingsrøret. </li><li> Pipette 500 pi vaskeopløsning på spin-filter. </li><li> Centrifuge ved 15.000 x g i 2 min. </li><li> Overfør spin filter over i en frisk opsamlingsrør. </li><li> Pipetter 50 pi elueringsopløsning opvarmettil 70 ° C på midten af ​​centrifugeringsfiltret. </li><li> Inkuber i 2-5 min ved 70 ° C. Centrifuger ved 15.000 x g i 1 min. </li><li> Pipettere yderligere 50 pi elueringsopløsning opvarmet til 70 ° C på midten af ​​centrifugeringsfiltret. </li><li> Gentag trin 1.2.2.1.18. </li></ol></li><li> Tilsæt 100 ug / ml glycogen, 0,1 volumen 3 M natriumacetat (pH 5,2), og 2,5 volumener absolut ethanol. Udfælde natten over ved -80 ° C. </li><li> Centrifuger ved 15.000 xg i 25 minutter ved 4 ° C. Supernatanten kasseres. </li><li> Vask pelleten med 1 ml 75% ethanol. Centrifuger ved 15.000 xg i 15 minutter ved 4 ° C. Fjern forsigtigt ethanol. </li><li> Der tørres i luft i 3-5 min. </li><li> Der tilsættes 10 pi af nuklease-frit vand til at opløse bundfaldet. </li><li> Opbevar ved -70 ° C. </li></ol></li></ol></li><li> RNA Kvalifikation og Kvantificering <ol><li> Ved hjælp af et spektrofotometer, fastlægge RNA koncentration ved noting absorbansen ved 260 nm og 280 nm. The A260 / A280-forholdet skal være 1,8-2,2. </li><li> Kontrollér kvaliteten RNA-prøven ved 0,8% agarosegelelektroforese 25. BEMÆRK: intakte eukaryote total RNA skal vise intense 28S og 18S rRNA bands. Forholdet mellem 28S versus 18S rRNA båndintensitet for en god RNA-præparat er ~ 2. </li></ol></li></ol> 2. Gel Elektroforese og overførsel BEMÆRK: buffer forberedelse protokoller er angivet i tabel 1. <ol><li> Formaldehyd Gel Fremstilling (1%) <ol><li> Skyl alt elektroforese udstyr med diethylpyrocarbonat (DEPC) vand. </li><li> Smelt 2,5 g agarose i 215 ml DEPC vand fuldstændigt i en mikrobølgeovn. </li><li> Tilføj 12,5 ml 10x 3- (N- morpholino) propansulfonsyre (MOPS) -buffer og 22,5 ml 37% formaldehyd. </li><li> Hæld det i elektroforese apparat med en 1,5 mm tyk kam. </li><li> Eliminer bobler eller skubbe them til kanterne af gelen med en ren kam. </li><li> Tillad gelen at størkne ved stuetemperatur. </li></ol></li><li> Prøvefremstilling <ol><li> Bland 1-10 ug totalt RNA og 11,3 pi prøvepuffer. </li><li> Bland 2 ug af RNA markør (1 ug / uL) med 11,3 pi af prøven buffer. </li><li> Tilføj nuklease-frit vand til prøverne fra 2.2.1 og 2.2.2 til et samlet volumen på 16 pi. </li><li> Der opvarmes til 60 ° C i 5 minutter, og derefter chill på is. </li><li> Bland 16 pi af prøven fra 2.2.3 med 4 pi sporing farvestof, som indeholder 0,1 pg / pL ethidiumbromid på is. ADVARSEL: Ethidiumbromid er muligvis teratogent og giftigt ved indånding. Læs ethidiumbromid sikkerhedsdatablad. </li></ol></li><li> Elektroforese <ol><li> 200 ml 10x MOPS buffer til 1800 ml Hedeselskabet 2 O at lave en 1x MOPS kører buffer. </li><li> Brug 1x MOPS løbebufferen at skylle brøndene i gelen. </li><li> Pre-run than gelere ved 20 V i 5 minutter. </li><li> Indlæse RNA-prøver i brøndene på gelen. </li><li> Dæk med folie for at undgå udsættelse for lys. Kørt ved 35 V i ca. 17 timer (natten over) </li><li> Undersøge og fotografere gel under et UV-lys. </li></ol></li><li> Overførsel <ol><li> Skær gelen for at fjerne den ubrugte del. </li><li> Forbered 500 ml 10x SSC puffer (250 ml 20x SSC buffer og 250 ml DEPC vand). </li><li> Vask gelen to gange med 10 x SSC puffer i 20 min under omrystning ved 50 rpm. </li><li> Cut 1 filterpapir ark store nok til at tjene som en væge papir, som absorberer transfer buffer fra overførslen bakke (figur 1). </li><li> Skær 4 stykker filterpapir til samme størrelse som gelen. </li><li> Cut 1 ark af positivt ladet nylonmembran til samme størrelse som gelen. </li><li> Markere et hak på gelen og membranen som markør for at sikre den korrekte orientering. </li><li> Soak membranen i 2x SSC buffer i 15 min. </li><li> Hæld 500 ml of 10x SSC buffer ind i transfer-bakken. </li><li> Læg store filter papirarket tværs af glaspladen og våd filtrerpapiret med overførselsbuffer. </li><li> Rul eventuelle bobler med en pipette. </li><li> Soak 2 stykker af pre-cut filter papir i transfer buffer og placere dem på midten af ​​filtrerpapiret fra trin 2.4.5. Fjern eventuelle bobler. </li><li> Læg gel top-side nedad på filterpapir. </li><li> Lægge membranen på toppen af ​​gelen. Juster hakkene i gelen og membranen. </li><li> Placer 2 stykker af pre-cut filtrerpapir på toppen af ​​membranen og befugte filtrerpapiret med overførselsbuffer. </li><li> Fjern eventuelle bobler mellem lagene. </li><li> Påfør plastikfolie omkring gelen for at sikre, at håndklæder kun opsuge buffer passerer gennem gelen og membranen. </li><li> Stable papirhåndklæder oven på filterpapir. </li><li> Placer en overdimensioneret glasplade oven på håndklæder. </li><li> Placer en vægt på toppen af ​​stakken. </li><li>Holde natten over for at tillade RNA at overføre fra gelen til membranen. </li></ol></li></ol> 3. Påvisning af N6 -methyladenosine <ol><li> Membrane Tværbinding <ol><li> Hold membranen fugtig i 2x SSC buffer. </li><li> Placer 2 ark filtrerpapir nedsænkes med 10x SSC buffer i UV tværbindingsmiddel. </li><li> Anbring membranen på toppen af ​​filtrerpapiret, med RNA-adsorberede opad. </li><li> Vælg &quot;autocrosslink mode&quot; (120.000 μJ) og tryk på knappen &quot;Start&quot; for at starte bestråling. </li><li> Undersøg membranen og gelen med en UV gel billede catcher at bekræfte RNA overførsel fra gelen til membranen. </li></ol></li><li> immunoblotting <ol><li> Blokere membranen i 5% fedtfri mælk i Tris-bufret saltvand med Tween 20 (TBST) buffer ved stuetemperatur i 1 time. </li><li> Vask tre gange med TBST-buffer i 15 min. </li><li> Inkuber membranen natten over i m 6 A (&lt;em&gt; N 6 -methyladenosine) antistof (1: 1000 i 5% fedtfri mælk TBST buffer) ved 4 ° C. </li><li> Vask membranen tre gange med TBST-buffer i 15 min. </li><li> Inkuber membranen i HRP-konjugeret æsel-anti-kanin-antistof (1: 2.000 i 5% fedtfri mælk TBST buffer) i 1 time ved stuetemperatur. </li><li> Vask membranen tre gange med TBST-buffer i 15 min. </li><li> Påfør forstærket kemiluminescerende substrat (0,125 ml pr cm2 af membranen). </li><li> Fange kemiluminescens med de optimale indstillinger for den digitale billeddanneren ifølge producentens anvisninger 26. </li></ol></li><li> m 6 A Kvantificering <ol><li> Mål den relative m 6 et kemiluminescerende intensitet ved hjælp af ImageJ software. <ol><li> Under software menuen ImageJ, skal du vælge &quot;File&quot; mulighed for at åbne den relevante fil. </li><li> Vælg &quot;Rektangel&quot; værktøj fra ImageJ og tegne en ramme omkringsignalerne. </li><li> Hvis ribosomalt RNA er ikke i fokus i forsøgene, undgå 18S og 28S ribosomale RNA m 6 A bånd til beregningen. </li><li> Klik på &quot;Command&quot; og &quot;1&quot; for at bekræfte den valgte vognbane. </li><li> Tryk på &quot;Command&quot; og &quot;3&quot; for at vise den valgte plot. </li><li> Klik på &quot;Straight&quot; værktøj og tegne linjer til at adskille det segmenterede område. </li><li> Klik på &quot;Wand&quot; værktøj til at registrere målingerne. </li><li> Eksportere data. </li><li> Normalisere niveauet af m 6 A med 18S ribosomale RNA-båndet fra ethidiumbromidfarvning. </li></ol></li></ol></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">
	<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.

Efter 14 dage i normal lys-mørke døgnrytmen fase blev de vildtypemus placeret i konstant mørke. RNA&#39;erne fra leveren blev samplet ved 4 timers intervaller og undersøgt med modificeret northern blotting. Methylering af rRNA&#39;er, mRNA&#39;er, og små RNA&#39;er blev klart påvist (figur 2). Sammenligningen mellem forskellige døgnrytmen tidspunkter (CT) kan præcist beregnes med 18S rRNA-standarden. Der var robust døgnrytmen oscillation af m 6 A niveauer i rRNA, mRNA, og små RNA. For at undgå interferens lille som mulig ved rRNA, kan polyadenylerede RNA&#39;er oprenses, som i trin 1.2.2. Efter oprensning kan rRNA stort set fjernes for at muliggøre bedre visualisering af andre RNA&#39;er (figur 3). <img alt="figur 1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html> g&gt; Figur 1: Samling af blot-overførselsenheden. Den kapillære overføringsenhed til blotting af RNA til membranen er vist. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">Klik her for at se en større version af dette tal.</a> <img alt="Figur 2" src="/files/ftp_upload/54672/54672fig2.jpg" /> Figur 2: døgnrytmen af m 6 A-niveauer i C57BL / 6J-mus. Denne figur viser m 6 En blot af totalt RNA fra lever fra vildtypemus aflivet på den første dag af den mørke-mørke fase efter 14 dage af en normal lys-mørke fase ved 4 timers intervaller. Kvantificeringen blev udført ved hjælp af m 6 A belysningen forskellen mellem 18S og 28S rRNA. Den m 6 En overflod blev normaliseret til 18S rRNA-båndet fra formaldehyd gel i bunden.et = &quot;_ blank&quot;&gt; Klik her for at se en større version af dette tal. <img alt="Figur 3" src="/files/ftp_upload/54672/54672fig3.jpg" /> Figur 3: m 6 A blots med eller uden rRNA. Repræsentative m 6 A blots af totalt RNA og polyadenyleret RNA fra lever fra vildtypemus er vist. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">Klik her for at se en større version af dette tal.</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, beskyttet mod 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 vand </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"> Der omrøres ved stuetemperatur </td><td></td><td></td><td></td></tr><tr><td> 2 </td><td colspan="3"> 20x SSC (pH 7,0) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Natriumchlorid </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 vand </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"> Der omrøres ved stuetemperatur, derefter autoklaveres </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 puffer </td><td> 500 </td><td> uL </td><td> 1x </td></tr><tr><td></td><td colspan="3"> Ficoll 400 </td><td> 0,75 </td><td> g </td><td> 15% </td></tr><tr><td></td><td colspan="3"> bromphenolblåt </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 vand </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"> Opbevares ved -20 ° C </td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> DEPC vand </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Fortynd ratio på 1: 1000 af DEPC i Hedeselskabet 2 O </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Omrør natten over ved stuetemperatur, derefter autoklaveres </td><td></td><td></td><td></td></tr><tr><td> 5 </td><td colspan="3"> Prøve buffer (beskytte mod lys) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10x MOPS puffer </td><td> 200 </td><td> uL </td><td></td></tr><tr><td></td><td colspan="3"> 37% formaldehyd </td><td> 270 </td><td> uL </td><td></td></tr><tr><td></td><td colspan="3"> formamid </td><td> 660 </td><td> uL </td><td></td></tr><tr><td></td><td colspan="3"> Opbevares ved -20 ° C </td><td></td><td></td><td></td></tr></tbody&gt; </table> Tabel 1: Buffere og opløsninger.

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 modificeret northern blotting metode til måling N6 -methyladenosine (m 6 A) ændringer i RNA er beskrevet. Den nuværende metode kan detektere ændringer i forskellige RNA og kontroller under forskellige eksperimentelle designs.

En metode til måling af RNA<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine Ændringer i celler og væv

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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