Name: a method for measuring rna n6-methyladenosine modifications in cells and tissues
Uploaded: 2016-12-05T13:00:00
Duration: 8 min 56 s
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).

Opmerking: Het totale RNA m 6 Een niveau omvat rRNA, mRNA, en andere kleine RNAs. Sinds ribosomaal RNA heeft overvloedige m 6 A wijzigingen, de meting van m 6 A levels moet dit feit te overwegen. 1. RNA Isolation <ol><li> Met behulp van de ribonuclease decontaminatie-oplossing (gespoten op papieren handdoeken), veeg de pipetten en de labtafel oppervlakken. Natte papieren handdoeken met nuclease-vrij water en veeg naar beneden pipetten en labtafel oppervlakken weer. </li><li> RNA-extractie <ol><li> Total RNA Isolation <ol><li> Met een bovenroerder, gehomogeniseerd 50-100 mg weefsel of 5-10 x 10 6 cellen in 1 ml RNA-isolatie oplossing gehandhaafd op 4 ° C. OPMERKING: More weefsel (100-150 mg) vereist voor vetweefsel monsters van muizen vanwege de lagere concentraties RNA daarin. Monsters afkomstig van muis hart, lever, skeletspier, long, hersenen en macrofagen zijn eerder gebruikt 4. </li><li> Incubate het monster homogenaten gedurende 5 minuten bij kamertemperatuur. </li><li> Voeg 100 ul van 1-broom-3-chloorpropaan (BCP) per 1 ml gehomogeniseerd. Schud de buizen krachtig gedurende 15 s en ga verder met totaal RNA te isoleren volgens de instructies van de fabrikant 24. </li></ol></li><li> Gepolyadenyleerde mRNA Zuivering van Geïsoleerde Totaal RNA <ol><li> Zuiveren gepolyadenyleerde mRNA met behulp van beschikbare kits of protocollen. <ol><li> Schud grondig resuspendeer elk van de volgende oplossingen: 2x bindoplossing, oligo (dT) polystyreen kralen en wasoplossing. Zorg ervoor dat de oligo (dT) kralen warm tot kamertemperatuur vóór gebruik. </li><li> Transfer 120 ul van elutieoplossing per preparaat in een buis en verhit tot 70 ° C in een verwarmingsblok. </li><li> Pipet tot 500 ug totaal RNA in een microcentrifugebuis. Met nuclease-vrij water, het volume aanpassen tot 250 ul. </li><li> Voeg 250 ul 2x binding oplossing van het totale RNA datlutie en vortex kort naar de inhoud te mengen. </li><li> Voeg 15 ul van oligo (dT) kralen en vortex grondig te mengen. </li><li> Incubeer het mengsel bij 70 ° C gedurende 3 min. </li><li> Verwijder het monster uit het verwarmingsblok en laten staan ​​bij kamertemperatuur gedurende 10 min. </li><li> Centrifugeer bij 15.000 xg gedurende 2 minuten. </li><li> Verwijder voorzichtig het supernatant, met achterlating van ongeveer 50 ul. </li><li> Voeg 500 ul van wassen oplossing voor resuspendeer de pellet door pipetteren. </li><li> Pipetteer de suspensie in een spin filter / collectie buis set. </li><li> Centrifugeer bij 15.000 xg gedurende 2 minuten. Verwijder de kolom verzamelbuis en gooi het doorstroomkanaal. Breng de kolom aan de collectie buis. </li><li> Pipetteer 500 ul van wassen oplossing op de spin-filter. </li><li> Centrifuge bij 15.000 x g gedurende 2 min. </li><li> Breng de spin-filter in een frisse collectie buis. </li><li> Pipetteer 50 ul van elutie verhittot 70 ° C op het midden van het rotatiefilter. </li><li> Incubeer gedurende 2-5 minuten bij 70 ° C. Centrifugeer bij 15.000 xg gedurende 1 min. </li><li> Pipetteer een extra 50 ui elutie-oplossing verwarmd tot 70 ° C op het midden van het rotatiefilter. </li><li> Herhaal stap 1.2.2.1.18. </li></ol></li><li> Voeg 100 ug / ml glycogeen, 0,1 volume 3 M natriumacetaat buffer (pH 5,2) en 2,5 volumes absolute ethanol. Precipiteren nacht bij -80 ° C. </li><li> Centrifugeer bij 15.000 xg gedurende 25 min bij 4 ° C. Verwijder het supernatant. </li><li> Was de pellet met 1 ml 75% ethanol. Centrifugeer bij 15.000 xg gedurende 15 min bij 4 ° C. Verwijder de ethanol voorzichtig. </li><li> Droge lucht gedurende 3-5 min. </li><li> Voeg 10 ul nuclease-vrij water aan de pellet lossen. </li><li> Bewaar bij -70 ° C. </li></ol></li></ol></li><li> RNA kwalificatie en kwantificering <ol><li> Met behulp van een spectrofotometer, bepalen de RNA concentratie kennisgevingng de absorptie bij 260 nm en 280 nm. De A260 / A280-verhouding moet 1,8-2,2. </li><li> Controleer de RNA-monster kwaliteit met behulp van 0,8% agarosegelelektroforese 25. LET OP: De intacte eukaryotische totaal RNA moet intense 28S en 18S rRNA bands laten zien. De verhouding van 28S versus 18S rRNA band intensiteit voor een goede voorbereiding is het RNA ~ 2. </li></ol></li></ol> 2. Gel Elektroforese en Transfer OPMERKING: De buffervoorbereiding protocollen zijn in tabel 1. <ol><li> Formaldehyde Gel Bereiding (1%) <ol><li> Spoel alle elektroforese-apparatuur met diethylpyrocarbonaat (DEPC) water. </li><li> Smelt 2,5 g agarose in 215 ml DEPC water volledig in de magnetron. </li><li> Voeg 12,5 ml 10x 3- (N- morfolino) propaansulfonzuur (MOPS) buffer en 22,5 ml 37% formaldehyde. </li><li> Giet het in de electroforese-inrichting met een 1,5 mm dik kam. </li><li> Elimineer bellen of duw tzoom aan de randen van de gel met een schone kam. </li><li> Laat de gel stollen bij kamertemperatuur. </li></ol></li><li> monstervoorbereiding <ol><li> Meng 1-10 ug totaal RNA en 11,3 pl monsterbuffer. </li><li> Meng 2 ug van het RNA marker (1 ug / ul) met 11,3 pl monsterbuffer. </li><li> Add nuclease-vrij water aan de monsters van 2.2.1 en 2.2.2 tot een totaal volume van 16 ul. </li><li> Verhit bij 60 ° C gedurende 5 minuten, en vervolgens relaxen op ijs. </li><li> Meng 16 pl van het monster 2.2.3 met 4 pl traceerkleurstof, die 0,1 ug bevat / ul ethidiumbromide op ijs. LET OP: Ethidiumbromide is mogelijk teratogeen en giftig bij inademing. Lees ethidiumbromide veiligheidsinformatieblad. </li></ol></li><li> elektroforese <ol><li> Voeg 200 ml 10x MOPS buffer tot 1.800 ml DDH 2 O tot een 1X MOPS loopbuffer te maken. </li><li> Gebruik de 1x MOPS loopbuffer aan de putjes van de gel te spoelen. </li><li> Pre-run tHij gel bij 20 V gedurende 5 min. </li><li> Laad de RNA monsters in de putjes van de gel. </li><li> Dek af met folie om blootstelling aan licht te voorkomen. Run op 35 V gedurende ongeveer 17 uur ( &#39;s nachts) </li><li> Onderzoeken en fotograferen van de gel onder een UV-licht. </li></ol></li><li> Overdracht <ol><li> Snijd de gel om het ongebruikte deel te verwijderen. </li><li> Bereid 500 ml 10 x SSC buffer (250 ml 20 x SSC buffer en 250 ml DEPC water). </li><li> Was de gel tweemaal met 10x SSC buffer gedurende 20 min met schudden bij 50 rpm. </li><li> Snijd 1 filtreerpapier vel groot genoeg om als een lont papier, dat transferbuffer opneemt uit de overdrachtschaalvorm (figuur 1). </li><li> Snijd 4 filtreerpapiertjes dezelfde grootte als de gel. </li><li> 1 gesneden vel positief geladen nylonmembraan dezelfde grootte als de gel. </li><li> Markeer een inkeping op de gel en het membraan als een marker om de juiste oriëntatie te verzekeren. </li><li> Geniet van het membraan in 2x SSC buffer gedurende 15 min. </li><li> Giet 500 ml of 10x SSC buffer in de overdracht lade. </li><li> Leg de grote filter vel papier over de glasplaat en bevochtig de filter papier met de overdracht buffer. </li><li> Uitrollen eventuele luchtbellen met een pipet. </li><li> 2 weken stukjes voorgesneden filtreerpapier in overdrachtsbuffer en plaats ze op het midden van het filterpapier uit stap 2.4.5. Verwijder eventuele luchtbellen. </li><li> Leg de gel top-kant naar beneden op het filter papier. </li><li> Lag het membraan bovenop de gel. Lijn de inkepingen van de gel en het membraan. </li><li> Leg 2 stukken van pre-cut filter papier op de top van het membraan en nat de filter papier met de overdracht buffer. </li><li> Verwijder eventuele luchtbellen tussen de lagen. </li><li> Toepassen plastic rond de gel dat de handdoeken alleen genieten buffer die door de gel en het membraan. </li><li> De stapel papieren handdoeken bovenop het filterpapier. </li><li> Plaats een grote glasplaat boven op de handdoeken. </li><li> Plaats een gewicht op de bovenkant van de stapel. </li><li>Blijf gedurende de nacht om het RNA om van de gel naar het membraan. </li></ol></li></ol> 3. Detectie van N 6 -methyladenosine <ol><li> membraan Crosslinking <ol><li> Houd het membraan vocht in 2x SSC buffer. </li><li> Plaats 2 vellen filterpapier ondergedompeld met 10x SSC buffer in UV crosslinker. </li><li> Plaats het membraan boven op het filterpapier, met de RNA-geadsorbeerde zijde naar boven. </li><li> Selecteer &quot;autocrosslink mode&quot; (120.000 uJ) en druk op de knop &quot;Start&quot; om de bestraling te starten. </li><li> Onderzoek de membraan en de gel met een UV-afbeelding gel catcher aan het RNA transfer van de gel aan het membraan te bevestigen. </li></ol></li><li> immunoblotting <ol><li> Blokkeer het membraan in 5% vetvrije melk in Tris-gebufferde zoutoplossing met Tween 20 (TBST) buffer bij kamertemperatuur gedurende 1 uur. </li><li> Was driemaal met TBST buffer gedurende 15 min. </li><li> Incubeer het membraan overnacht in m 6 A (&lt;em&gt; N 6 -methyladenosine) antilichaamoplossing (1: 1000 in 5% vetvrije melk TBST buffer) bij 4 ° C. </li><li> Was het membraan driemaal met TBST buffer gedurende 15 min. </li><li> Incubeer het membraan in het HRP-geconjugeerd ezel anti-konijn antilichaam-oplossing (1: 2000 in 5% vetvrije melk TBST buffer) gedurende 1 uur bij kamertemperatuur geroerd. </li><li> Was het membraan driemaal met TBST buffer gedurende 15 min. </li><li> Breng het versterkt chemiluminescent substraat (0,125 ml per cm2 membraan). </li><li> Leg de chemiluminescentie met de optimale instellingen voor de digitale beeldvormer volgens de instructies van de fabrikant 26. </li></ol></li><li> m 6 een kwantificering <ol><li> Meet de relatieve m 6 een chemiluminescentie-intensiteit met de ImageJ software. <ol><li> Onder de ImageJ software menu de optie &quot;File&quot; om het relevante bestand te openen. </li><li> Selecteer de &quot;rechthoek&quot; tool van ImageJ en trek een kader rondde signalen. </li><li> Als ribosomaal RNA is niet de focus van de experimenten, vermijd de 18S en 28S ribosomale RNA m 6 A banden voor de berekening. </li><li> Klik &quot;Commando&quot; en &quot;1&quot; om de geselecteerde baan te bevestigen. </li><li> Druk op &quot;Command&quot; en &quot;3&quot; om de geselecteerde plot tonen. </li><li> Klik op de &quot;Straight&quot; tool en lijnen te vestigen op de gesegmenteerde te scheiden. </li><li> Klik op de &quot;Wand&quot; tool om de metingen te nemen. </li><li> De gegevens exporteren. </li><li> Normaliseren van de niveaus van m 6 A met de 18S ribosomale RNA band uit ethidium bromide kleuring. </li></ol></li></ol></li></ol>

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De auteurs hebben niets te onthullen.

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.

Na 14 dagen in normaal licht-donker circadiane fase werden de wild-type muizen geplaatst in constante duisternis. De RNA&#39;s uit de lever werden bemonsterd op 4 uur intervallen en studeerde met gemodificeerde Noord-blotting. De methylering van rRNA, mRNA&#39;s en kleine RNA duidelijk gedetecteerd (figuur 2). De vergelijking tussen verschillende circadiane tijden (CT) kan nauwkeurig worden berekend met de 18S rRNA standaard. Er was sterke circadiane oscillatie van m 6 A niveaus in rRNA, mRNA, en kleine RNAs. Om de storing aangeboden door rRNA te voorkomen, kan gepolyadenyleerde RNA&#39;s worden gezuiverd, zoals in stap 1.2.2. Na zuivering, kan het rRNA grotendeels geëlimineerd om een betere visualisatie van andere RNA&#39;s (figuur 3). <img alt="Figuur 1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html> g&gt; Figuur 1: Montage van de vlek transporteenheid. De capillaire blotting overbrengeenheid voor het RNA aan het membraan getoond. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">Klik hier om een grotere versie van deze figuur te bekijken.</a> <img alt="Figuur 2" src="/files/ftp_upload/54672/54672fig2.jpg" /> Figuur 2: circadiane ritme van de m 6 A levels in C57BL / 6J muizen. Deze figuur toont de m 6 Een blot van totaal RNA van levers van wild-type muizen geofferd op de eerste dag van de donkere donkere fase na 14 dagen van een normaal licht-donker fase bij 4 uur intervallen. De kwantificering werd gedaan met behulp van de m 6 beeld Een dichtheidsverschil tussen 18S en 28S rRNA. De m 6 een overvloed werd genormaliseerd naar het 18S rRNA band uit de formaldehyde gel op de bodem.et = &quot;_ blank&quot;&gt; Klik hier om een ​​grotere versie van deze figuur te bekijken. <img alt="figuur 3" src="/files/ftp_upload/54672/54672fig3.jpg" /> Figuur 3: De m 6 Een blots met of zonder rRNA. Representatieve m 6 Een blots van totaal RNA en gepolyadenyleerd RNA uit de lever van wildtype muizen worden getoond. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">Klik hier om een grotere versie van deze figuur te bekijken.</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, bescherming tegen licht) </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"> Natriumacetaat </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"> DEPC water </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Totaal </td><td> 1 </td><td> L </td><td></td></tr><tr><td></td><td colspan="3"> Roer bij kamertemperatuur </td><td></td><td></td><td></td></tr><tr><td> 2 </td><td colspan="3"> 20x SSC-buffer (pH 7,0) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> natriumchloride </td><td> 175.3 </td><td> g </td><td> 3 M </td></tr><tr><td></td><td colspan="3"> trinatriumcitraat </td><td> 88.3 </td><td> g </td><td> 0,3 M </td></tr><tr><td></td><td colspan="3"> DEPC water </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Totaal </td><td> 1 </td><td> L </td><td></td&gt; </tr><tr><td></td><td colspan="3"> Roer bij kamertemperatuur, vervolgens autoclaveren </td><td></td><td></td><td></td></tr><tr><td> 3 </td><td colspan="3"> Tracking Dye </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10x MOPS buffer </td><td> 500 </td><td> pl </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"> broomfenolblauw </td><td> 0.01 </td><td> g </td><td> 0,2% </td></tr><tr><td></td><td colspan="3"> xyleencyanol </td><td> 0.01 </td><td> g </td><td> 0,2% </td></tr><tr><td></td><td colspan="3"> DEPC water </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Totaal </td><td> 5 </td><td> mL </td><td></td></tr><tr><td></td><td colspan="3"> Bewaren bij -20 ° C </td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> DEPC water </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Verdun ratio 1: 1000 in met DEPC DDH 2 O </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Roer overnacht bij kamertemperatuur, vervolgens autoclaveren </td><td></td><td></td><td></td></tr><tr><td> 5 </td><td colspan="3"> Monsterbuffer (bescherming tegen licht) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10x MOPS buffer </td><td> 200 </td><td> pl </td><td></td></tr><tr><td></td><td colspan="3"> 37% formaldehyde </td><td> 270 </td><td> pl </td><td></td></tr><tr><td></td><td colspan="3"> formamide </td><td> 660 </td><td> pl </td><td></td></tr><tr><td></td><td colspan="3"> Bewaren bij -20 ° C </td><td></td><td></td><td></td></tr></tbody&gt; </table> Tabel 1: Buffers en oplossingen.

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

Gemodificeerde Northern blotting methode voor het meten N6 -methyladenosine (m 6 A) veranderingen in RNA wordt beschreven. De huidige methode kan wijzigingen in diverse RNA&#39;s en controles onder verschillende experimentele ontwerpen te detecteren.

Een methode voor het meten van RNA<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine Wijzigingen in cellen en weefsels

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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