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. 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. 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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.

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	<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, ...

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

Research

JoVE Journal

Biochemistry

8.4K Views.  Simon Fraser University. 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.

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

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.

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.

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

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 ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...