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-Biot...

<ol>
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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. 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=Crystal+Structures+of+the+Mango-II+RNA+Aptamer+Reveal+Heterogeneous+Fluorophore+Binding+and+Guide+Engineering+of+Variants+with+Improved+Selectivity+and+Brightness.">Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness.</a> Biochemistry. 57, 3544-3548 (2018).</li><li>Trachman, R., 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=Mango-III+is+a+compact+fluorogenic+RNA+aptamer+of+unusual+structural+complexity.">Mango-III is a compact fluorogenic RNA aptamer of unusual structural complexity.</a> Nature Chemical Biology. , (2018).</li><li>Panchapakesan, S. S. S., Jeng, S. C. Y., 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=RNA+complex+purification+using+high-affinity+fluorescent+RNA+aptamer+tags.">RNA complex purification using high-affinity fluorescent RNA aptamer tags.</a> Annals of the New York Academy of Sciences. , (2015).</li><li>Filonov, G. S., Kam, C. W., Song, W., Jaffrey, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-gel+imaging+of+RNA+processing+using+Broccoli+reveals+optimal+aptamer+expression+strategies.">In-gel imaging of RNA processing using Broccoli reveals optimal aptamer expression strategies.</a> Chemistry &amp; Biology. 22, 649-660 (2015).</li><li>Milligan, J. F., Groebe, D. R., Witherell, G. W., Uhlenbeck, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligoribonucleotide+synthesis+using+T7+RNA+polymerase+and+synthetic+DNA+templates.">Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.</a> Nucleic Acids Research. 15, 8783-8798 (1987).</li><li>A Typical DNase I Reaction Protocol (M0303). NEB Available from: <a target="_blank" href="https://www.neb.com/protocols/1/01/01/a-typical-dnase-i-reaction-protocol-m0303">https://www.neb.com/protocols/1/01/01/a-typical-dnase-i-reaction-protocol-m0303</a> (2018)</li><li>Sambrook, J., Russell, D. 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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 border="0" cellpadding="0" cellspacing="0" style="width:924px;" width="924">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">0.8mm Thick Comb 14 Wells for 30 mL PAGE gels</td>
			<td style="width:192px;">LabRepCo</td>
			<td style="width:147px;">11956042</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">101-1000 &#181;L&#160; tips</td>
			<td>Fisher</td>
			<td>02-707-511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20-200 &#181;L low retention&#160; tips</td>
			<td>Fisher Scientific</td>
			<td>02-717-143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrylamide:N,N&#39;-methylenebisacrylamide (40% 19:1)</td>
			<td>Bioreagents</td>
			<td>BP1406-1</td>
			<td>Acute toxicity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrylamide:N,N&#39;-methylenebisacrylamide (40% 29:1)</td>
			<td>Fisher</td>
			<td>BP1408-1</td>
			<td>Acute toxicity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Anachemia</td>
			<td>02116-380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminium backed TLC plate</td>
			<td>Sigma-Aldrich</td>
			<td>1164840001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amersham Imager 600</td>
			<td>GE Healthcare Lifesciences</td>
			<td>29083461</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Persulfate</td>
			<td>Biorad</td>
			<td>161-0700</td>
			<td>Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BL21 cells</td>
			<td>NEB</td>
			<td>C2527H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric Acid</td>
			<td>ACP</td>
			<td>B-2940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol Blue sodium salt</td>
			<td>Sigma</td>
			<td>B8026-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloloform</td>
			<td>ACP</td>
			<td>C3300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol</td>
			<td>Sigma Aldrich Alcohols</td>
			<td>D0632-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>ThermoFisher</td>
			<td>EN0525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA Disodium Salt</td>
			<td>ACP</td>
			<td>E-4320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Commerial&#160;</td>
			<td>P016EAAN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flat Gel Loading tips</td>
			<td>Costar</td>
			<td>CS004854</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formamide 99%</td>
			<td>Alfa Aesar</td>
			<td>A11076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel apparatus set with spacers and combs</td>
			<td>LabRepCo</td>
			<td>41077017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Dish with Plastic lid</td>
			<td>Pyrex</td>
			<td>1122963</td>
			<td style="width:275px;">Should be large enough to fit your gel piece</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Anachemia</td>
			<td>43567-540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Anachemia</td>
			<td>464140468</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageQuanTL</td>
			<td>GE Healthcare Lifesciences</td>
			<td>29000605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG</td>
			<td>Invitrogen</td>
			<td>15529-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>ACP</td>
			<td>P-2940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Caledron</td>
			<td>4903-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M3409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>ACP</td>
			<td>S-2830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td>BDH</td>
			<td>BDH9292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orbital Rotator</td>
			<td>Lab-Line</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol</td>
			<td>Invitrogen</td>
			<td>15513-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round Gel Loading tips</td>
			<td>Costar</td>
			<td>CS004853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate dibasic</td>
			<td>Caledron</td>
			<td>8120-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate monobasic</td>
			<td>Caledron</td>
			<td>8180-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBRGold</td>
			<td>ThermoFisher</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 RNA Polymerase</td>
			<td>ABM</td>
			<td>E041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T7024-50 ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TO1-3PEG-Biotin Fluorophore</td>
			<td>ABM</td>
			<td>G955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Fisher</td>
			<td>BP152-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>Fisher</td>
			<td>BP1421-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Sigma</td>
			<td>P9496-100</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Urea</td>
			<td>Fisher</td>
			<td>U15-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene Cyanol</td>
			<td>Sigma</td>
			<td>X4126-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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, ...

The Mango-tagged gel imaging methodology demonstrated here is robust and is anticipated to be able to be simply extended in terms of sensitivity and specificity. This would further simplify the routine purification of biologically important RNAs and RNA complexes by the simple expedient of adding a Mango-tag to the RNA of interest. RNAs are being implicated in more and more diseases therefor this post dating method is an easy and fast way to be able to track and visualize RNA.Extra care will be required in transferring the gel from one place to another as the gels are fragile and prone to breaking. To prepare a 30 milliliter denaturing gel, first select the polyacrylamide percentage with the appropriate Bromophenol blue, and xylene cyanol dyes compared to an RNA of interest to insure high band separation. Then, mix solutions A, B, and C according to table one and add APS and timud.Immediately pour the gel solution into an appropriate gel casting apparatus. Insert the desired cone and leave the gel to polymerase for approximately 30 minutes. Make up the gel tank using 1 XTBE solution and carefully remove the cone.Carefully lift up the gel and mount on to the gel apparatus and secure using binder clips. Using a syringe, aspirate out the wells immediately prior to sample loading. Prepare 1X RNA samples by adding 2X denaturing gel loading solution to the RNA samples of interest.Use a thermocycler to heat the samples at 95 degrees Celsius for five minutes. Then, load cooled samples to the bottom of each well using gel loading tips. And well the gel at room temperature ensuring the power is effectively low to not crack the glass plate of the gel apparatus.Prepare 1X gel staining solution according to the manuscript in a clean glass container that is wide enough to comfortably fit the gel. Add enough 1X gel staining solution to the container so that the gel is completely covered with the solution when it is placed on an orbital rotator. Once the gel finishes running, remove the gel from the apparatus, cut off its'wells, and a corner of the gel in order to help determine the orientation.Then, carefully transfer the gel into the 1X gel staining solution. To perform the imaging, carefully decant the staining solution and rinse the gel quickly with water. Finally, carefully transfer the gel onto the tray to be placed in the imager.Roll a glass pipette over the gel to remove trapped bubbles and excess liquid. And proceed to taking a gel image. Mango aptamers one, two, three, four were substantially resist to denaturation up to approximately one molar Urea concentration.20 to 40 minute staining time was sufficient for maximum gel florescence. Longer time was not suitable for small RNAs used in this study as they started to diffuse out of the gel. Fluorometer analysis showed that Mango aptamers one, two, and three in the presence of Urea could fold more rapidly than aptamer four.In the absence of Urea, folding was much more rapid as was expected. The sensitivity of the post staining method was shown by single bands corresponding to well folded RNAs for each of the Mango variants in native and denaturing gels with a slightly less sensitivity for the later one. Quantification of native gels was log linear over about one order of magnitude with Mango one, two, and four behaving in a more linear fashion than Mango three.Quantification of denaturing gels were more linear suggesting that the presence of Urea in the denaturing gel might provide a more homogenous way to fold the aptamers once they are placed in the TO1 biotin staining solution. Mango tagged RNAs can be detected in the presence of total RNA using TO1 biotin staining compared to SG staining. Remember to make sure that the gel staining container is large enough to fit the gel otherwise the gel may fold on to itself.The RNA samples will transfer from one place to another. Similar to the denaturing gels, this method can be performed for a native gel. Run the gel in a cold room at a lower wattage to maintain native conditions.

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Research

JoVE Journal

Biochemistry

8.4K 次观看.  Simon Fraser University. 此处演示的芒果标记凝胶成像方法非常坚固，预计在灵敏度和特异性方面可以简单地扩展。这将进一步简化生物重要RNA和RNA复合物的常规纯化，只需在感兴趣的RNA中加入芒果标签的简单权宜之计。RNA 被牵连到越来越多的疾病中，因为这种后约会方法是一种能够跟踪和可视化RNA的简便而快速的方法。将凝胶从一个地方转移到另一个地方时需要格外小心，因为凝胶很脆弱，...