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.
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.
