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).

NOTE: The total RNA m6A level includes rRNA, mRNA, and other small RNAs. Since ribosomal RNA has abundant m6A modifications, the measurement of m6A levels will need to consider this fact.
1. RNA Isolation
<ol>
	<li>Using the ribonuclease decontamination solution (sprayed onto paper towels), wipe the pipettes and lab bench surfaces. Wet paper towels with nuclease-free water and wipe down pipettes and lab bench surfaces again.</li>
	<li>RNA Extraction
	<ol>
		<li>Total RNA Isolation
		<ol>
			<li>Using an overhead stirrer, homogenize 50-100 mg of tissue or 5-10 x 106 cells in 1 mL of RNA isolation solution maintained at 4 &#176;C. 
			NOTE: More tissue (100-150 mg) may be required for adipose tissue samples from mice because of the lower RNA concentrations therein. Samples sourced from mouse heart, liver, skeletal muscle, lung, brain, and macrophages have been employed previously4.</li>
			<li>Incubate the sample homogenates for 5 min at room temperature.</li>
			<li>Add 100 &#181;L of 1-bromo-3-chloropropane (BCP) per 1 mL of sample homogenate. Shake the tubes vigorously for 15 s and proceed to isolate total RNA according to the manufacturer&#39;s instructions24.</li>
		</ol>
		</li>
		<li>Polyadenylated mRNA Purification from Isolated Total RNA
		<ol>
			<li>Purify polyadenylated mRNA using available kits or protocols.
			<ol>
				<li>Shake thoroughly to re-suspend each of the following solutions: 2x binding solution, oligo(dT) polystyrene beads, and wash solution. Ensure that the oligo(dT) beads warm to room temperature before use.</li>
				<li>Transfer 120 &#181;L of elution solution per preparation into a tube and heat to 70 &#176;C in a heating block.</li>
				<li>Pipette up to 500 &#181;g of total RNA into a microcentrifuge tube. With nuclease-free water, adjust the volume to 250 &#181;L.</li>
				<li>Add 250 &#181;L of 2x binding solution to the total RNA solution and vortex briefly to mix the contents.</li>
				<li>Add 15 &#181;L of oligo(dT) beads and vortex thoroughly to mix.</li>
				<li>Incubate the mixture at 70 &#176;C for 3 min.</li>
				<li>Remove the sample from the heating block and let it stand at room temperature for 10 min.</li>
				<li>Centrifuge at 15,000 x g for 2 min.</li>
				<li>Carefully remove the supernatant, leaving behind approximately 50 &#181;L.</li>
				<li>Add 500 &#181;L of wash solution to re-suspend the pellet by pipetting.</li>
				<li>Pipette the suspension into a spin filter/collection tube set.</li>
				<li>Centrifuge at 15,000 x g for 2 min. Remove the column from collection tube and discard the flow-through. Return the column to the collection tube.</li>
				<li>Pipette 500 &#181;L of wash solution onto the spin filter.</li>
				<li>Centrifuge at 15,000 x g for 2 min.</li>
				<li>Transfer the spin filter into a fresh collection tube.</li>
				<li>Pipette 50 &#181;L of elution solution heated to 70 &#176;C onto the center of the spin filter.</li>
				<li>Incubate for 2-5 min at 70 &#176;C. Centrifuge at 15,000 x g for 1 min.</li>
				<li>Pipette an additional 50 &#181;L of elution solution heated to 70 &#176;C onto the center of the spin filter.</li>
				<li>Repeat Step 1.2.2.1.18.</li>
			</ol>
			</li>
			<li>Add 100 &#181;g/mL glycogen, 0.1 volume of 3 M sodium acetate buffer (pH 5.2), and 2.5 volumes of absolute ethanol. Precipitate overnight at -80 &#176;C.</li>
			<li>Centrifuge at 15,000 x g for 25 min at 4 &#176;C. Discard the supernatant.</li>
			<li>Wash the pellet with 1 mL of 75% ethanol. Centrifuge at 15,000 x g for 15 min at 4 &#176;C. Carefully remove the ethanol.</li>
			<li>Dry in air for 3-5 min.</li>
			<li>Add 10 &#181;L of nuclease-free water to dissolve the pellet.</li>
			<li>Store at -70 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>RNA Qualification and Quantification
	<ol>
		<li>Using a spectrophotometer, determine the RNA concentration by noting the absorbance at 260 nm and 280 nm. The A260/A280 ratio should be 1.8-2.2.</li>
		<li>Check the RNA sample quality using 0.8% agarose gel electrophoresis25. 
		NOTE: The intact eukaryotic total RNA should show intense 28S and 18S rRNA bands. The ratio of 28S versus 18S rRNA band intensity for a good RNA preparation is ~2.</li>
	</ol>
	</li>
</ol>
2. Gel Electrophoresis and Transfer
NOTE: The buffer preparation protocols are given in Table 1.
<ol>
	<li>Formaldehyde Gel Preparation (1%)
	<ol>
		<li>Rinse all electrophoresis equipment with diethylpyrocarbonate (DEPC) water.</li>
		<li>Melt 2.5 g of agarose in 215 mL of DEPC water completely in a microwave oven.</li>
		<li>Add 12.5 mL of 10x 3-(N-morpholino)propanesulfonic acid (MOPS) buffer and 22.5 mL of 37% formaldehyde.</li>
		<li>Pour it into the electrophoresis apparatus with a 1.5 mm thick comb.</li>
		<li>Eliminate bubbles or push them to the edges of the gel with a clean comb.</li>
		<li>Allow the gel to solidify at room temperature.</li>
	</ol>
	</li>
	<li>Sample Preparation
	<ol>
		<li>Mix 1-10 &#181;g of total RNA and 11.3 &#181;L of sample buffer.</li>
		<li>Mix 2 &#181;g of the RNA marker (1 &#181;g/&#181;L) with 11.3 &#181;L of the sample buffer.</li>
		<li>Add nuclease-free water to the samples from 2.2.1 and 2.2.2 to a total volume of 16 &#181;L.</li>
		<li>Heat at 60 &#176;C for 5 min, and then chill on ice.</li>
		<li>Mix 16 &#956;L of the sample from 2.2.3 with 4 &#956;L of tracking dye, which contains 0.1 &#956;g/&#956;L ethidium bromide on ice. 
		CAUTION: Ethidium bromide is possibly teratogenic and toxic if inhaled. Read ethidium bromide safety datasheet.</li>
	</ol>
	</li>
	<li>Electrophoresis
	<ol>
		<li>Add 200 mL of 10x MOPS buffer to 1,800 mL of ddH2O to make a 1x MOPS running buffer.</li>
		<li>Use the 1x MOPS running buffer to rinse the wells of the gel.</li>
		<li>Pre-run the gel at 20 V for 5 min.</li>
		<li>Load the RNA samples into the wells of the gel.</li>
		<li>Cover with foil to avoid light exposure. Run at 35 V for about 17 h (overnight)</li>
		<li>Examine and photograph the gel under a UV light.</li>
	</ol>
	</li>
	<li>Transfer
	<ol>
		<li>Cut the gel to remove the unused portion.</li>
		<li>Prepare 500 mL of 10x SSC buffer (250 mL of 20x SSC buffer and 250 mL of DEPC water).</li>
		<li>Wash the gel twice with 10x SSC buffer for 20 min with shaking at 50 rpm.</li>
		<li>Cut 1 filter paper sheet large enough to serve as a wick paper, which absorbs transfer buffer from the transfer tray (Figure 1).</li>
		<li>Cut 4 pieces of filter paper to the same size as the gel.</li>
		<li>Cut 1 sheet of positively-charged nylon membrane to the same size as the gel.</li>
		<li>Mark a notch on the gel and the membrane as a marker to ensure the proper orientation.</li>
		<li>Soak the membrane in 2x SSC buffer for 15 min.</li>
		<li>Pour 500 mL of 10x SSC buffer into the transfer tray.</li>
		<li>Lay the large filter paper sheet across the glass plate and wet the filter paper with transfer buffer.</li>
		<li>Roll out any bubbles with a pipette.</li>
		<li>Soak 2 pieces of pre-cut filter paper in transfer buffer and place them on the center of the filter paper from step 2.4.5. Remove any bubbles.</li>
		<li>Lay the gel top-side down on the filter paper.</li>
		<li>Lay the membrane on top of the gel. Align the notches of the gel and the membrane.</li>
		<li>Place 2 pieces of pre-cut filter paper on top of the membrane and wet the filter paper with transfer buffer.</li>
		<li>Remove any bubbles between the layers.</li>
		<li>Apply plastic wrap around the gel to ensure that the towels only soak up buffer passing through the gel and the membrane.</li>
		<li>Stack the paper towels on top of the filter paper.</li>
		<li>Place an oversized glass plate on top of the towels.</li>
		<li>Place a weight on the top of the stack.</li>
		<li>Keep overnight to allow the RNA to transfer from the gel to the membrane.</li>
	</ol>
	</li>
</ol>
3. Detection of N6-methyladenosine
<ol>
	<li>Membrane Crosslinking
	<ol>
		<li>Keep the membrane damp in 2x SSC buffer.</li>
		<li>Place 2 sheets of filter paper immersed with 10x SSC buffer into UV crosslinker.</li>
		<li>Place the membrane on top of the filter paper, with the RNA-adsorbed side facing up.</li>
		<li>Select &#34;autocrosslink mode&#34; (120,000 &#181;J) and press the &#34;Start&#34; button to initiate the irradiation.</li>
		<li>Examine the membrane and the gel with a UV gel image catcher to confirm the RNA transfer from the gel to the membrane.</li>
	</ol>
	</li>
	<li>Immunoblotting
	<ol>
		<li>Block the membrane in 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) buffer at room temperature for 1 hr.</li>
		<li>Wash thrice with TBST buffer for 15 min.</li>
		<li>Incubate the membrane overnight in m6A (N6-methyladenosine) antibody solution (1:1,000 in 5% non-fat milk TBST buffer) at 4 &#176;C.</li>
		<li>Wash the membrane thrice with TBST buffer for 15 min.</li>
		<li>Incubate the membrane in the HRP-conjugated donkey anti-rabbit antibody solution (1:2,000 in 5% non-fat milk TBST buffer) for 1 h at room temperature.</li>
		<li>Wash the membrane thrice with TBST buffer for 15 min.</li>
		<li>Apply the enhanced chemiluminescent substrate (0.125 mL per cm2 of membrane).</li>
		<li>Capture the chemiluminescence with the optimum settings of the digital imager, according to manufacturer&#39;s instructions26.</li>
	</ol>
	</li>
	<li>m6A Quantification
	<ol>
		<li>Measure the relative m6A chemiluminescent intensity using the ImageJ software.
		<ol>
			<li>Under the ImageJ software menu, select the &#34;File&#34; option to open the pertinent file.</li>
			<li>Select the &#34;Rectangle&#34; tool from ImageJ and draw a frame around the signals.</li>
			<li>If ribosomal RNA is not the focus of the experiments, avoid the 18S and 28S ribosomal RNA m6A bands for the calculation.</li>
			<li>Click &#34;Command&#34; and &#34;1&#34; to confirm the selected lane.</li>
			<li>Press &#34;Command&#34; and &#34;3&#34; to show the selected plot.</li>
			<li>Click the &#34;Straight&#34; tool and draw lines to separate the segmented area.</li>
			<li>Click the &#34;Wand&#34; tool to record the measurements.</li>
			<li>Export the data.</li>
			<li>Normalize the levels of m6A with the 18S ribosomal RNA band from the ethidium bromide staining.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

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

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

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

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

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

After 14 days in normal light-dark circadian phase, the wild-type mice were placed in constant darkness. The RNAs from the liver were sampled at 4 hr intervals and studied with modified northern blotting. The methylation of rRNAs, mRNAs, and small RNAs were clearly detected (Figure 2). The comparison between different circadian times (CT) can be accurately calculated with the 18S rRNA standard. There was robust circadian oscillation of m6A levels in rRNA, mRNA, and small RNAs.
To avoid the interference proffered by rRNA, polyadenylated RNAs can be purified, as in step 1.2.2. After purification, the rRNA can be largely eliminated to allow for better visualization of other RNAs (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/54672/54672fig1.jpg" /> 
Figure 1: Assembling the blot transfer unit. The capillary transfer unit for blotting the RNA to the membrane is shown. <a href="http://cloudflare.jove.com/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54672/54672fig2.jpg" /> 
Figure 2: Circadian rhythm of the m6A levels in C57BL/6J mice. This figure shows the m6A blot of total RNA from livers of wild-type mice sacrificed on the first day of the dark-dark phase after 14 days of a normal light-dark phase at 4 h intervals. The quantification was done using the m6A image density difference between 18S and 28S rRNA. The m6A abundance was normalized to the 18S rRNA band from the formaldehyde gel at the bottom. <a href="http://cloudflare.jove.com/files/ftp_upload/54672/54672fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54672/54672fig3.jpg" /> 
Figure 3: The m6A blots with or without rRNA. Representative m6A blots of total RNA and polyadenylated RNA from the livers of wild-type mice are shown. <a href="http://cloudflare.jove.com/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</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, protect from light)</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">Sodium acetate</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">Total</td>
			<td>1</td>
			<td>L</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Stir at room temperature</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">Sodium chloride</td>
			<td>175.3</td>
			<td>g</td>
			<td>3 M</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Trisodium citrate</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">Total</td>
			<td>1</td>
			<td>L</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Stir at room temperature, then autoclave</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>&#956;L</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">Bromophenol blue</td>
			<td>0.01</td>
			<td>g</td>
			<td>0.2%</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Xylene Cyanol</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">Total</td>
			<td>5</td>
			<td>mL</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Store at -20 &#176;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">Dilute ratio at 1:1,000 of DEPC in ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Stir overnight at room temperature, then autoclave</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="3">Sample buffer (protect from light)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">10x MOPS buffer</td>
			<td>200</td>
			<td>&#956;L</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">37% formaldehyde</td>
			<td>270</td>
			<td>&#956;L</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Formamide</td>
			<td>660</td>
			<td>&#956;L</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Store at -20 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Buffers and solutions.

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

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

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 ...

a-method-for-measuring-rna-n6-methyladenosine-modifications-cells

Research

JoVE Journal

Biochemistry

10.9K Views.  Chang Gung Memorial Hospital, Chang Gung University College of Medicine.  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's en controles onder verschillende experimentele ontwerpen te detecteren. 

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

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

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

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

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

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

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

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

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

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

Genetics

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

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

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

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

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

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

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

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

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

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

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

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

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

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

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

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

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

Cancer Research

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

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

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

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

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

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

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

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

Developmental Biology

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

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

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

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

Chemistry

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

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

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