The authors thank Razvan Cojocaru and Amir Abdolahzadeh for their technical assistance and Lena Dolgosheina for proofreading the manuscript. Funding was provided for this project by a Canadian Natural Sciences and Engineering Research Council (NSERC) operating grant to P.J.U.

1. Preparation of the reagents
<ol>
	<li>TO1-Biotin poststaining solution (gel staining solution)
	<ol>
		<li>Make 1 L of 1 M phosphate buffer at pH 7.2 at 25 &#176;C by adding 342 mL of 1 M of Na2HPO4 and 158 mL of 1 M NaH2PO4. Adjust pH to 7.2 at 50 mM by adding the appropriate phosphate solution. Sterile-filter using a 0.2 &#181;m filter and store the solution in plasticware at room temperature.</li>
		<li>Prepare 5x gel staining solution (without TO1-Biotin) as follows. Make up a 1 L solution by mixing 247.5 mL of ddH2O, 700 mL of 1 M KCl, 50 mL of 1 M phosphate buffer (pH 7.2), and 2.5 mL of Tween 20. Sterile-filter using a 0.2 &#181;m filter and store the solution in plasticware. It can be stored at room temperature. 
		NOTE: MgCl2 can be added to this solution if required as it can potentially stabilize RNA complexes. This will have a modest impact on the fluorescent signal2.</li>
		<li>Make up a 1x gel staining solution using the 5x gel staining solution from step 1.1.2 and, immediately prior to use, supplement it with TO1-Biotin fluorophore (see Table of Materials) to a final concentration of 20 nM. For example, add 20 mL of 5x gel staining solution to 80 mL of deionized water to make 100 mL of 1x gel staining solution, and add 2 &#181;L of a 1 mM TO1-Biotin stock (prepared in dimethylformamide). 
		NOTE: The extinction coefficient for the TO1-Biotin dye at 500 nm is 63,000 M-1&#183;cm-1, measured in staining solution1.</li>
	</ol>
	</li>
	<li>Denaturing PAGE (2x denaturing gel loading solution and solutions A, B, C, ammonium persulfate, and tetramethylethylenediamine)
	<ol>
		<li>Prepare 50 mL of 2x denaturing gel loading solution by mixing 40 mL of formamide, 0.5 mL of 0.5 M ethylenediaminetetraacetic acid (EDTA, pH 8.0), and 9.5 mL ddH2O. The solution can be stored at room temperature. Loading dyes are not added to this solution as it may obscure fluorescent imaging.</li>
		<li>Prepare 2x denaturing gel loading dye as follows. When purifying RNA and DNA oligonucleotides, add 0.5 mL of 2.5% (w/v) bromophenol blue (BB) and 0.5 mL of 2.5% (w/v) xylene cyanol (XC) to the solution described in step 1.2.1, and add 8.5 mL of ddH2O instead of 9.5 mL. The solution can be stored at room temperature.</li>
		<li>Prepare 100 mL of solution A by weighing out 200.2 g of urea (formula weight: 60.06 g/mol) and adding it to 312.5 mL of 40% 19:1 acrylamide:N,N&#39;-methylenebisacrylamide. Stir it with a magnetic stir bar at maximum speed until dissolved (about 3 h) and make up the solution to 500 mL using ddH2O. Note that the final concentrations are 6.667 M urea and 25% 19:1 acrylamide:N,N&#39;-methylenebisacrylamide. Store at 4 &#176;C in clean plasticware.</li>
		<li>Prepare 1 L of solution B by weighing out 400.4 g of urea and make up the solution to 1 L with ddH2O; stir until the urea has dissolved. Solution B can be kept at room temperature.</li>
		<li>Prepare solution C (10x Tris-borate-EDTA [TBE]) as follows. Prepare 4 L of 10x TBE by weighing out 432 g of Tris base and 220 g of boric acid, adding 160 mL of 0.5 M EDTA with a pH of 8 (filtered), and making up the solution to 4 L with ddH2O. A large stock of this buffer is made as it is also used as gel running buffer.</li>
		<li>Prepare 10% ammonium persulfate (APS) by dissolving 1 g of APS into a total volume of 10 mL of ddH2O. Store at 4 &#176;C.</li>
		<li>Keep tetramethylethylenediamine (TEMED) handy. Store it at 4 &#176;C together with the APS solution.</li>
	</ol>
	</li>
	<li>Native PAGE (2x native gel loading solution)
	<ol>
		<li>Prepare 50 mL of 2x native gel loading solution by mixing 25 mL of 100% glycerol, 10 mL of 5x gel staining solution, and 15 mL of ddH2O to make up the solution to 50 mL. 
		NOTE: As in the denaturing gel section, loading dyes can be added to this stock, but their addition can potentially obscure the fluorescent signal in the gel and, thus, should be avoided.</li>
	</ol>
	</li>
</ol>
2. Preparation and loading of denaturing gels
<ol>
	<li>Prepare a denaturing gel with an appropriate percentage by following Table 1. Consider the specific gel casting system; 30 mL gels are commonly used. The appropriate gel percentage can be estimated by using Table 2: select the polyacrylamide percentage where the BB and XC dyes have motilities faster and slower, respectively, than the RNA of interest, to ensure high-band separation in the relevant size range.</li>
	<li>Mix solutions A, B, and C according to Table 1 and add APS and TEMED immediately prior to pouring the gel. Mix the solutions well in clean plasticware.</li>
	<li>Pour the gel solution into an appropriate gel casting apparatus, after ensuring that all components are scrupulously clean. Lift one side of the apparatus so that the gel is slightly tilted when pouring, to avoid any air bubbles becoming trapped within the gel itself.</li>
	<li>Insert the desired comb and leave it to polymerize (approximately 30 min). Observe the polymerization by looking closely for a change in the index of refraction around the gel wells. Make up the gel tank by using 1x solution C (1x TBE) and carefully remove the comb. Using a syringe, aspirate out the wells immediately prior to sample loading.</li>
	<li>Prepare denaturing samples as follows. Add 2x denaturing gel loading solution to the RNA samples of interest to make the denaturing gel loading solution 1x and heat-denature at 95 &#176;C for 5 min by using a thermocycler or water bath.</li>
	<li>Prior to loading the samples, air-cool them several minutes until they are cool to the touch. Load the samples by layering them on the bottom of each well, using gel-loading tips. 
	NOTE: Round tips can be used for gels of 1 mm thickness or more; use flat tips for thinner gels.</li>
	<li>Run the denaturing gels at room temperature. Ensure that the wattage is sufficiently low so that the glass plates of the gel system do not crack. 
	NOTE: In our laboratory, 28 W for 20 cm x 16 cm plates was sufficient for this purpose.</li>
</ol>
3. Preparation and loading of native gels
<ol>
	<li>Prepare a native gel with an appropriate percentage by referring to Table 3. Consider the specific gel casting system, but keep in mind that 30 mL gels are commonly used. Mix the solutions of 1x TBE, 40% 29:1 acrylamide:N,N&#8217;-methylenebisacrylamide, and glycerol according to Table 3, and add APS and TEMED immediately prior to pouring the gel. Proceed as described in steps 2.3 and 2.4.</li>
	<li>Prepare native samples by adding 2x native gel loading solution to the RNA samples of interest to make the solution 1x and allow it to incubate at room temperature for 100 min prior to running the gel to ensure complete RNA folding.</li>
	<li>Run the native gel in a 4 &#176;C cold room, ensuring that the wattage is sufficiently low to not heat the gel. 
	NOTE: In our laboratory, 14 W for 20 cm x 16 cm plates was sufficient for this purpose.</li>
</ol>
4. RNA Preparation by run-off T7 transcription
NOTE: DNA sequences used for the run-off transcription10 of RNA Mango constructs were ordered commercially. In this method, DNA oligonucleotides containing the reverse complement (RC) of both the sequence to be transcribed and the T7 promoter are hybridized to a T7 promoter top strand sequence and then transcribed in vitro. Below, for each oligonucleotide, the RC of the Mango core sequence is shown in bold and the RC of the T7 promoter region is shown in italics. Residues in regular font correspond to otherwise arbitrary complementary helical regions required to allow the Mango core to properly fold.
Mango I: GCA CGT ACT CTC CTC TCC GCA CCG TCC CTT CGT ACG TGC CTA TAG TGA GTC GTA TTA AAG 
Mango II: GCA CGT ACT CTC CTC TTC CTC TCC TCT CCT CGT ACG TGC CTA TAG TGA GTC GTA TTA AAG 
Mango III: GGC ACG TAC GAA TAT ACC ACA TAC CAA TCC TTC CTT CGT ACG TGC CTA TAG TGA GTC GTA TTA AAG 
Mango IV: GCA CGT ACT CGC CTC ATC CTC ACC ACT CCC TCG GTA CGT GCC TAT AGT GAG TCG TAT TAA AG 
T7 Top Strand: CTT TAA TAC GAC TCA CTA TAG G
<ol>
	<li>Preparative gel purification of DNA oligonucleotides
	<ol>
		<li>For a 0.2 &#181;mol-scale DNA synthesis, resuspend the deprotected DNA oligonucleotide in 100 &#181;L of ddH2O and 100 &#181;L of 2x denaturing loading dye (from step 1.2.2). In this case, including gel loading dyes in the loading solution at the same concentration as in step 1.2.2 is preferred.</li>
		<li>Purify the DNA oligonucleotide using a 50 mL preparative scale denaturing gel of the appropriate polyacrylamide percentage; use Table 2 to choose the gel percentage. For example, use an 8% gel for a 50 nt sequence. Ideally, bromophenol blue should run faster than the oligonucleotide, and xylene cyanol should run slower. 
		NOTE: In our laboratory, such gels were cast using casting spacers that were 1.5 mm thick and a gel-loading comb with wells that were 2&#8211;2.5 cm wide.</li>
		<li>Load 100 &#181;L of the DNA oligonucleotide solutions prepared in step 4.1.2 per preparative gel well as described in steps 2.4&#8211;2.7.</li>
		<li>Carefully dry the outside of the gel, remove the glass plates, and cover both sides of the gel in plastic wrap. Place it onto a fluorescent imaging screen (aluminum-backed thin-layer chromatography plates impregnated with fluorophore are an economical solution) and use a short-wavelength UV hand-held lamp to visualize the DNA bands by UV shadowing.</li>
		<li>A crisp, well-defined shadow should be observed if the DNA synthesis is of high quality. Mark the bands on the plastic wrap, using a permanent marker. 
		NOTE: Protect skin and eyes from UV light and keep exposures short to avoid damaging the nucleic acid sample.</li>
		<li>Placing the gel on a clean glass plate, carefully cut out the marked bands and place each gel fragment in 400 &#181;L of 300 mM NaCl. Elute the DNA overnight, using a rotator at room temperature.</li>
		<li>Recover the eluent in a clean centrifuge tube and add 2.5 equivalents of ethanol to precipitate the DNA. Vortex well and place the tube at -20 &#176;C for 30 min.</li>
		<li>Pellet the sample in a bench top centrifuge at 156,000 x g for 30 min at 4 &#176;C. Carefully remove the supernatant and resuspend the pellet in ddH2O. Use a spectrophotometer to accurately determine the DNA oligonucleotide concentration. Store the sample as a 10 &#956;M stock for convenience at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Run-off transcription and gel purification of RNA samples
	<ol>
		<li>Prepare 5x nucleoside triphosphate (NTP) stock by mixing liquid NTP stocks made up of a final concentration of 40 mM guanosine triphosphate (GTP, 11,400 M-1&#183;cm-1), 25 mM cytidine triphosphate (CTP, 7,600 M-1&#183;cm-1), 25 mM adenosine triphosphate (ATP, 5,000 M-1&#183;cm-1), and 10 mM uridine triphosphate (UTP, 10,000 M-1&#183;cm-1); all extinction coefficients at 260 nm. 
		NOTE: Liquid or powdered stocks can be obtained commercially. If preparing primary stocks from powder, carefully adjust the pH to a final pH of 7.9, using 1 M NaOH. Aliquot the stock in 1.5 mL microcentrifuge tubes and store it at -20 &#176;C.</li>
		<li>Prepare 100 mL of 10x T7 transcription buffer stock by mixing 25.6 mL of 1 M Tris-HCl, 14.4 mL of 1 M Tris base, 26 mL of 1 M MgCl2, 10 mL of 10% Triton X-100, and 0.637 g of spermidine. Make up the stock to a final volume of 100 mL by adding ddH2O. 
		NOTE: A final pH of 7.9 of the 1x solution should be confirmed using a calibrated pH meter. The 10x should be sterile-filtered and stored at -20 &#176;C in suitably sized aliquots.</li>
		<li>Perform transcription as follows.
		<ol>
			<li>Add the following reagents to make up a final concentration of 1x T7 transcription buffer using the 10x T7 transcription buffer stock and ddH2O (from step 4.2.2), 1x NTPs, 10 mM of dithiothreitol (DTT), 1 &#956;M T7 top strand sequence (see section 4 Note for the sequence), 1 &#956;M gel-purified Mango DNA sequence (step 2.1), and T7 RNA polymerase enzyme (1 U/&#181;L).</li>
			<li>Vortex, spin down, and incubate the solution at 37 &#176;C for 2 h or until it becomes cloudy and a white precipitate forms at the bottom of the tube. A 50 &#181;L transcription should result in 50 &#181;L of ~50 &#181;M RNA after gel purification. Add an equal volume of 2x denaturing dye and store it at -20 &#176;C until ready for gel purification.</li>
		</ol>
		</li>
		<li>Gel-purify the resulting RNA as described in the DNA oligonucleotides gel purification section by following steps 4.1.2&#8210;4.1.8, substituting the RNA sample for the DNA. 
		NOTE: RNA is extremely sensitive to RNase degradation, so ensure that in all steps, gloves and a clean lab coat are worn at all times. Ensure all samples are prepared and stored in single-use plasticware to protect against RNase contamination, and wash glass plates carefully with hot soap and water prior to use, rinse them thoroughly with ddH2O, and dry the glassware with the assistance of ethanol applied from a squeeze bottle.</li>
		<li>Use 1 &#956;L of the final RNA sample and, using a droplet-based spectrophotometer, determine the absorbance at 260 nm. Using an extinction coefficient determined by the nearest neighbor method, calculate the RNA concentration and adjust the sample concentration to 10 &#956;M with ddH2O for convenience. Store the RNA samples at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Escherichia coli total crude nucleic acid extraction 
	NOTE: The following protocol is an example. This protocol uses endogenously expressed Mango I-tagged 6S RNA from a plasmid in E. coli, and induction from this plasmid is described elsewhere in detail4.
	<ol>
		<li>For the purposes of this study, prepare 500 mL of induced cells for both the pEcoli-RNA Mango and the pEcoli-T1 plasmids (the cells are induced at an OD600nm of 1). Pellet the cells and store them at -80 &#176;C prior to use.</li>
		<li>Perform RNA extraction as follows.
		<ol>
			<li>Take 0.5 mL of the induced pEcoli cell pellet and add 500 &#956;L of equilibrated phenol. Then, vortex the sample; the solution will become milky white. Centrifuge at maximum speed for 2 min to separate the layers.</li>
			<li>Extract the top layer, which will be slightly yellow, and repeat step 4.3.2.1 by adding an equal volume of phenol, centrifuging, and extracting for a total of 5x (or until the middle layer, which is opaque white, goes away and only two clear layers are left).</li>
			<li>Add the phenol-extracted aqueous volume to an equal volume of chloroform, vortex, and then, centrifuge at maximum speed for 2 min.</li>
			<li>Extract the aqueous layer and add NaCl to a final concentration of 300 mM. Precipitate the resulting nucleic acid by the addition of 2.5 equivalents of ethanol, vortex, spin down, and precipitate at -20 &#176;C for at least 30 min.</li>
			<li>Pellet in a bench top centrifuge at 15.6 x 1,000 x g at 4 &#176;C for 30 min. Carefully remove the supernatant and resuspend the pellet in 100 &#956;L of ddH2O.</li>
			<li>Add a DNase I digestion step to this procedure, following the referenced protocol11, to obtain total RNA. 
			NOTE: Vortex vigorously till the pellet is completely dissolved in ddH2O.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Post-gel staining
<ol>
	<li>Prepare 1x gel staining solution as per the recipe in step 1.1.3 and add it to a clean glass container that is wide enough to comfortably fit the gel. 
	NOTE: Borosilicate glass containers with snap fit lids serve this purpose well.</li>
	<li>Add enough 1x gel staining solution to the container so that the gel is completely covered with the solution and the liquid sloshes over the top of the gel when it is placed on the orbital rotator. 
	NOTE: The container must be big enough to fit the gel so that it can move around and have enough buffer in the container to fully cover the gel.</li>
	<li>Once the native or denaturing gel has finished running (section 2 or 3, respectively), remove the gel from the apparatus and cut off its wells. It can be useful to remove a corner of the gel for orientation later in the analysis.</li>
	<li>Carefully transfer the gel to the 1x gel staining solution prepared in step 5.2. 
	NOTE: The gels are fragile and prone to breaking so be careful when transferring the gel. Keep the lid on the staining container at all times so as not contaminate the gel.</li>
	<li>Place the gel on an orbital rotator at a speed of 100 rpm for 30 min at room temperature. 
	NOTE: Make sure that the gel does not fold back onto itself; otherwise, the RNA may diffuse out of the gel and so label a different part of the gel.</li>
</ol>
6. Imaging Mango-tagged RNAs in gel
<ol>
	<li>Carefully decant the imaging buffer. Rinse the gel quickly with water, ensuring enough liquid remains to keep the gel slightly mobile in the container.</li>
	<li>Carefully transfer the gel onto the imager. Transfer by carefully picking up the sides of the gel and placing it onto the imager tray; alternatively, the gel can be slowly poured onto the tray. Make sure there are no bubbles underneath the gel and that there is no excess liquid under the gel. A pipette rolled over the gel can be useful to remove any excess liquid. 
	NOTE: Use a paper towel to soak up the excess fluid.</li>
	<li>Take a gel image, observing fluorescence between excitation wavelength 510 nm and emission wavelength 535 nm (for example, green light at 520 nm wavelength fluorescence settings on the imager). Make sure the settings are fully optimized on the instrument and carefully follow the instructions for the instrument used.</li>
</ol>

<ol>
	<li>Dolgosheina, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Mango+Aptamer-Fluorophore:+A+Bright,+High-Affinity+Complex+for+RNA+Labeling+and+Tracking.">RNA Mango Aptamer-Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking.</a> ACS Chemical Biology. , (2014).</li><li>Autour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorogenic+RNA+Mango+aptamers+for+imaging+small+non-coding+RNAs+in+mammalian+cells.">Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.</a> Nature Communications. 9, 656 (2018).</li><li>Dolgosheina, E. V., Unrau, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorophore-binding+RNA+aptamers+and+their+applications:+Fluorophore-binding+RNA+aptamers.">Fluorophore-binding RNA aptamers and their applications: Fluorophore-binding RNA aptamers.</a> Wiley Interdisciplinary Reviews: RNA. , (2016).</li><li>Panchapakesan, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonucleoprotein+Purification+and+Characterization+using+RNA+Mango.">Ribonucleoprotein Purification and Characterization using RNA Mango.</a> RNA. , 1592-1599 (2017).</li><li>Trachman, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+high-affinity+fluorophore+binding+and+activation+by+RNA+Mango.">Structural basis for high-affinity fluorophore binding and activation by RNA Mango.</a> Nature Chemical Biology. 13, 807 (2017).</li><li>Trachman, R. 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A patent is pending on the Mango fluorogenic system.

A significant advantage of the Mango fluorescent tag is that a single tag can be used in multiple ways. The high brightness and affinity of these aptamers make them useful not only for in cell visualization2 but also for in vitro RNA or RNP purification4. Therefore, gel imaging extends the versatility of the Mango tag in a straightforward way. Mango gel imaging sensitivity is slightly less than that of a northern blot14 but can easily detect 60&#8211...

Mango is an RNA tagging system consisting of a set of four small fluorescent RNA aptamers that bind tightly (nanomolar binding) to simple derivatives of the thiazole-orange (TO1-Biotin, Figure 1A)1,2,3. Upon binding, the fluorescence of this ligand is increased 1,000- to 4,000-fold depending on the specific aptamer. The high brightness of the Mango system, which for Mango III exceeds that of enhanced green fluorescent protein (eGFP), combined with the nanomolar binding affinity of the RNA Mango aptamers, allows it to be used both in the imaging and the purification of RNA complexes2,4.
The X-ray structures of Mango I5, II6, and III7 have been determined to high resolution, and all three aptamers utilize an RNA quadruplex to bind TO1-Biotin (Figure 1B&#8211;D). The compact cores of all three aptamers are isolated from the external RNA sequence via compact adaptor motifs. Mango I and II both utilize a flexible GNRA-like loop adaptor to connect their Mango cores to an arbitrary RNA duplex (Figure 1B,C). In contrast, Mango III uses a rigid triplex motif to connect its core to an arbitrary RNA helix (Figure 1D, purple residues), while the structure of Mango IV is not currently known. As the ligand-binding core of each of these aptamers is separated from the external RNA sequence by these helical adaptors, it appears likely that they can all be simply incorporated into a variety of RNAs. The bacterial 6S regulatory RNA (Mango I), components of the yeast spliceosome (Mango I), and the human 5S RNA, U6 RNA, and a C/D scaRNA (Mango II and IV) have all been successfully tagged in this fashion2,8, suggesting that many biological RNAs can be tagged using the RNA Mango aptamer system.
Denaturing and native gels are commonly used to study RNAs. Denaturing gels are often used to judge RNA size or RNA processing, but typically, in the case of a northern blot, for example, require several slow and sequential steps in order to generate an image. While other RNA fluorogenic aptamers, such as RNA Spinach and Broccoli, have been used successfully for gel imaging9, no fluorogenic aptamer system to date possesses the high brightness and affinity of the Mango system, making it of considerable interest to investigate Mango&#8217;s gel-imaging abilities. In this study, we wondered if the RNA Mango system could be simply extended to gel imaging, as the excitation and emission wavelengths of TO1-Biotin (510 nm and 535 nm, respectively) are appropriate for imaging in the eGFP channel common to most fluorescent gel-scanning instrumentation.
The post-gel staining protocol presented here provides a rapid way to specifically detect Mango-tagged RNA molecules in native and denaturing polyacrylamide gel electrophoresis (PAGE) gels. This staining method involves soaking gels in a buffer containing potassium and TO1-Biotin. RNA Mango aptamers are G-quadruplex based and potassium is required to stabilize such structures. Using RNA transcribed from minimal Mango-encoding DNA templates (see the protocol section), we can simply detect as little as little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels, using a straightforward staining protocol. In contrast to common nonspecific nucleic acid stains (see Table of Materials, referred to SG from hereon), we can clearly identify Mango-tagged RNA even when high concentrations of total untagged RNA are present in the sample.

<table><tbody><tr><td>0.8mm Thick Comb 14 Wells for 30 mL PAGE gels</td><td>LabRepCo</td><td>11956042</td><td></td></tr><tr><td>101-1000 &amp;micro;L&amp;nbsp; tips</td><td>Fisher</td><td>02-707-511</td><td></td></tr><tr><td>20-200 &amp;micro;L low retention&amp;nbsp; tips</td><td>Fisher Scientific</td><td>02-717-143</td><td></td></tr><tr><td>Acrylamide:N,N&#039;-methylenebisacrylamide (40% 19:1)</td><td>Bioreagents</td><td>BP1406-1</td><td>Acute toxicity</td></tr><tr><td>Acrylamide:N,N&#039;-methylenebisacrylamide (40% 29:1)</td><td>Fisher</td><td>BP1408-1</td><td>Acute toxicity</td></tr><tr><td>Agar</td><td>Anachemia</td><td>02116-380</td><td></td></tr><tr><td>Aluminium backed TLC plate</td><td>Sigma-Aldrich</td><td>1164840001</td><td></td></tr><tr><td>Amersham Imager 600</td><td>GE Healthcare Lifesciences</td><td>29083461</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Biorad</td><td>161-0700</td><td>Harmful</td></tr><tr><td>BL21 cells</td><td>NEB</td><td>C2527H</td><td></td></tr><tr><td>Boric Acid</td><td>ACP</td><td>B-2940</td><td></td></tr><tr><td>Bromophenol Blue sodium salt</td><td>Sigma</td><td>B8026-25G</td><td></td></tr><tr><td>Chloloform</td><td>ACP</td><td>C3300</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma Aldrich Alcohols</td><td>D0632-5G</td><td></td></tr><tr><td>DNase I</td><td>ThermoFisher</td><td>EN0525</td><td></td></tr><tr><td>EDTA Disodium Salt</td><td>ACP</td><td>E-4320</td><td></td></tr><tr><td>Ethanol</td><td>Commerial&amp;nbsp;</td><td>P016EAAN</td><td></td></tr><tr><td>Flat Gel Loading tips</td><td>Costar</td><td>CS004854</td><td></td></tr><tr><td>Formamide 99%</td><td>Alfa Aesar</td><td>A11076</td><td></td></tr><tr><td>Gel apparatus set with spacers and combs</td><td>LabRepCo</td><td>41077017</td><td></td></tr><tr><td>Glass Dish with Plastic lid</td><td>Pyrex</td><td>1122963</td><td>Should be large enough to fit your gel piece</td></tr><tr><td>Glycerol</td><td>Anachemia</td><td>43567-540</td><td></td></tr><tr><td>HCl</td><td>Anachemia</td><td>464140468</td><td></td></tr><tr><td>ImageQuanTL</td><td>GE Healthcare Lifesciences</td><td>29000605</td><td></td></tr><tr><td>IPTG</td><td>Invitrogen</td><td>15529-019</td><td></td></tr><tr><td>KCl</td><td>ACP</td><td>P-2940</td><td></td></tr><tr><td>MgCl2</td><td>Caledron</td><td>4903-01</td><td></td></tr><tr><td>MgSO4</td><td>Sigma-Aldrich</td><td>M3409</td><td></td></tr><tr><td>NaCl</td><td>ACP</td><td>S-2830</td><td></td></tr><tr><td>NaOH</td><td>BDH</td><td>BDH9292</td><td></td></tr><tr><td>Orbital Rotator</td><td>Lab-Line</td><td></td><td></td></tr><tr><td>Phenol</td><td>Invitrogen</td><td>15513-039</td><td></td></tr><tr><td>Round Gel Loading tips</td><td>Costar</td><td>CS004853</td><td></td></tr><tr><td>Sodium Phosphate dibasic</td><td>Caledron</td><td>8120-1</td><td></td></tr><tr><td>Sodium Phosphate monobasic</td><td>Caledron</td><td>8180-01</td><td></td></tr><tr><td>SYBRGold</td><td>ThermoFisher</td><td>S11494</td><td></td></tr><tr><td>T7 RNA Polymerase</td><td>ABM</td><td>E041</td><td></td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>T7024-50 ml</td><td></td></tr><tr><td>TO1-3PEG-Biotin Fluorophore</td><td>ABM</td><td>G955</td><td></td></tr><tr><td>Tris Base</td><td>Fisher</td><td>BP152-500</td><td></td></tr><tr><td>Tryptone</td><td>Fisher</td><td>BP1421-500</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P9496-100</td><td></td></tr><tr><td>Urea</td><td>Fisher</td><td>U15-3</td><td></td></tr><tr><td>Xylene Cyanol</td><td>Sigma</td><td>X4126-10G</td><td></td></tr><tr><td>Yeast Extract</td><td>Bioshop</td><td>YEX401.500</td><td></td></tr></tbody></table>

fluorescent visualization of mango-tagged rna in polyacrylamide gels via a poststaining method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Short Mango-tagged RNAs were prepared as described in the protocol section. Assuming that fluorescence in denaturing conditions would be most difficult to observe owing to the presence of urea in the gels, we first studied the resistance of the Mango aptamers to urea, which acts as a nucleic acid denaturant. We found that Mango aptamers are substantially resistant to denaturation up to a urea concentration of approximately 1 M (Figure 2A). Prior to adding gel staining sol...

Watch this Scientific Journal Video about Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method at JoVE.com

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

fluorescent-visualization-mango-tagged-rna-polyacrylamide-gels-via

Research

JoVE Journal

Biochemistry

8.5K Views.  Simon Fraser University. De Mango-tagged gel imaging methodologie die hier is aangetoond is robuust en zal naar verwachting eenvoudig kunnen worden uitgebreid in termen van gevoeligheid en specificiteit. Dit zou de routinematige zuivering van biologisch belangrijke RNAs- en RNA-complexen verder vereenvoudigen door eenvoudig een Mango-tag toe te voegen aan het RNA van belang. RNAs worden betrokken bij meer en meer ziekten daarom deze post dating methode is een gemakkelijke en snelle manier om te kunnen volgen en visua...

Video: Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.0 &#181;M) SYBR Green I (SG I) and 0.20 mM nitro blue tetrazolium that produces a purple precipitate of formazan when exposed to sunlight or specifically blue light. Also, DNA recovery tests were performed using an ampicillin resistant plasmid in an agarose gel stained with our method. A larger number of colonies was obtained with our method than with traditional staining using SG I with ultraviolet illumination. The described method is fast, specific, and non-toxic for DNA detection, allowing visualization of biomolecules to the "naked eye" without a transilluminator, and is inexpensive and appropriate for field use. For these reasons, our new DNA staining method has potential benefits to both research and industry.

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

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

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biology

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that detect levels of metabolites and signaling molecules. In order to study dynamic changes in each of these systems, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro and cellular turn-on kinetics for fluorogenic RNA aptamers using a plate reader equipped with a sample injector and a flow cytometer, respectively. We show that the in vitro kinetics for the fluorescence activation of the Spinach2 and Broccoli aptamers can be modeled as two-phase association reactions and have differing fast phase rate constants of 0.56 s&#8722;1 and 0.35 s&#8722;1, respectively. In addition, we show that the cellular kinetics for the fluorescence activation of Spinach2 in Escherichia coli, which is further limited by dye diffusion into the Gram-negative bacteria, is still sufficiently rapid to enable accurate sampling frequency on the minute timescale. These methods to analyze fluorescence activation kinetics are applicable to other fluorogenic RNA aptamers that have been developed.

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

The protocol presents two methods to determine the kinetics of the fluorogenic RNA aptamers Spinach2 and Broccoli. The first method describes how to measure fluorogenic aptamer kinetics in vitro with a plate reader, while the second method details the measurement of fluorogenic aptamer kinetics in cells by flow cytometry.

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that ...

Dual-Color End-Labeling and Vesicle Encapsulation of RNAs for smFRET-TIRF Studies

Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their conformational changes over time. To monitor RNA dynamics with smFRET, we developed a method to covalently label RNAs at their termini with a FRET pair of fluorophores. This direct end-labeling strategy targets the 5&#39;-phosphate by carbodiimide (EDC)/N-hydroxysuccinimide (NHS) activation and the 3&#39;-ribose by periodate oxidation, which can be adapted to other RNAs regardless of their size and sequence to study them independently of artificial modifications. Furthermore, the 5&#39;-EDC/NHS activation is of general interest to all nucleic acids with a 5&#39;-phosphate. The use of commercially available chemicals eliminates the need to synthesize RNA-specific probes.
Total Internal Reflection Fluorescence (TIRF) microscopy requires the surface-immobilized molecules of interest to be within the evanescent field to be illuminated. A sophisticated way of keeping the RNA molecules within the evanescent field is to encapsulate them in phospholipid vesicles. Encapsulation benefits from the best of both worlds, tethering the molecule to the surface while enabling free diffusion of the molecule. We ensure that each vesicle contains only a single RNA molecule, enabling single-molecule imaging. Upon dual-end labeling and encapsulation of the RNA of interest, smFRET measurements offer a dynamic and detailed view of RNA behavior.

This article describes the dual-color labeling of long RNAs at termini positions and their surface immobilization via encapsulation in phospholipid vesicles for single-molecule FRET TIRF microscopy applications. Combining these techniques enables precise visualization and analysis of RNA dynamics at the single-molecule level.

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Denaturing Urea Polyacrylamide Gel Electrophoresis for RNA Analysis: A Technique to Separate Fluorescently Labeled Phosphorylated RNA Oligonucleotides from their Non-Phosphorylated Equivalents

Source: Pillon, M. C., et al., <a href="https://www.jove.com/v/61258">Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates</a>. J. Vis. Exp. (2020)
In this video, we demonstrate a denaturing polyacrylamide gel electrophoresis for the separation of phosphorylated RNA species from their non-phosphorylated counterparts. The RNA species are fluorescently labeled for laser detection on the gel.

Source: Pillon, M. C., et al., Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates. J. Vis. Exp. (2020)
In this ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

Fluorescent Silver Staining: A Technique to Visualize Total Protein in Polyacrylamide Gels

After electrophoresis, submerge the gel in a 100-milliliter solution containing ethanol and acetic acid on an orbital shaker, and incubate twice for 30 minutes each, at room temperature while shaking at 50 RPM. Next, wash the gel three times with ultrapure water in a clean container, with each wash lasting 10 minutes.
First, dissolve 0.01 grams of silver nitrate in 10 milliliters of ultrapure water to prepare a stock solution with a concentration of 0.1%. Add 100 microliters of this stock solution into 100 milliliters of ultrapure water to make the silver nitrate working solution. In a fume hood, submerge the gel in 100 milliliters of the silver nitrate working solution in a sealed glass chamber.
Use aluminum foil to protect the gel from light during impregnation, and incubate for 1 hour while shaking at 50 RPM on an orbital shaker. After this, wash the gel twice with ultrapure water in a clean container, with each wash using approximately 100 milliliters of water and lasting 60 seconds.
It is important to use ultrapure water to clean the gel after the silver impregnation step to minimize background staining.
First, add 50 milliliters of ultrapure water to 3 milligrams of TPE-4TA dye. Sonicate the solution for 3 minutes, and add 5 microliters of 1 molar sodium hydroxide solution in between each sonication session, to help dissolve the dye, usually up to three times. Then, check the fluorescence of the solution under a 365-nanometer UV lamp to ensure that the dye is fully dissolved. Only weak or non-emissive solutions indicate full dissolution.
To prepare the fluorogenic developing solution, add 10 milliliters of the TPE-4TA stock solution to 90 milliliters of ultrapure water. Use a pH meter to check the pH of the solution. Tune the solution to a pH between 7 and 9, using diluted sodium hydroxide solution or acetic acid, if the pH is out of range.
Next, transfer the gel to a clean and sealable container with 100 milliliters of the fluorogenic developing solution, and ensure that the gel is completely immersed. Seal the container, and cover it to protect it from light. Shake the container overnight on an orbital shaker at 50 RPM and at room temperature.
Transfer the gel to a clean container, and de-stain it in 100 milliliters of 10% ethanol for 30 minutes. Then, rinse the gel in ultrapure water for 5 minutes. The gel can be visualized on a benchtop trans-illuminator, or imaged on a gel documentation machine at the 365-nanometer channel or the 302-nanometer channel.

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

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