Moore:RNA Gels

General

Your RNA will be put into an RNA loading dye that is primarily formamide (brand name FORMAzol, from MRC).The RNA will be heated in that sample buffer, so you want the pH controlled (and relatively low) and EDTA present to make sure there's no free divalent cation when heated.

We commonly use bis-Tris-Cl as a buffer for RNA samples at pH 6.5, you can prepare a small stock (~50 mL) at 250 mM, adjust pH to 6.5 with HCl and store separately for use in many solutions (like having a Tris, MOPS, HEPES, or acetate buffer stock on hand). It's a good idea to keep preservatives all over the place - bacteria can grow in the most awful conditions and secrete RNase (and protease). So, spiking your bis-Tris stock and anything else freshly made with 0.1 mM EDTA is a good precaution (from a prepared, pH ~7-7.5 adjusted 250 mM Na-EDTA stock). Just remember to take the EDTA amount into consideration when making buffers that require free divalent cations.*Smoore (talk) 08:59, 9 March 2020 (PDT):

For the loading dye:

The dye in the sample is bromophenol blue (BPB). You can make a 1% stock in water, it may appear a bit orange/yellow if the pH is low, but will be OK once added to final dye mix. The amount you add is up to you, some folks like a lighter sample buffer, some a darker one. The sample buffer will be ~1-1.5X, so you can take it easy with the dye amount.

In an attempt to maximize the formamide concentration in the final sample, try to make a 95% formamide solution, with ~10 mM buffer, EDTA, and some BPB. It will end up something like: ~4.8 mL of formamide, 200 uL bis-Tris stock, 20 uL EDTA stock, and some BPB to taste. Remove an aliquot of the final mix into a 1.5 mL microfuge tube for easy/rack access (also saves most in case of spill of accidental contamination). Record what you made, each person ends up with it a bit different, but can be useful for troubleshooting bad gels).

There is a pretty broad range of RNA you can load, 0.2-2 ug total RNA per well is common, it depends on your well size, the nature of the RNA, and the gel thickness. Try 0.5 ug/well to start with the Bio-Rad mini system using a 10-well comb. You want to maximize the fraction of denaturing formamide, but still add a controllable/accurate amount of RNA for even loading. Also, make enough sample for several gels. For example, if your RNA sample is 1.0 mg/mL (1.0 ug/uL), mix 10 uL RNA sample with 90 of loading dye, that will make it 0.1 ug/uL. You can then load 5-10 uL of that in a well.

You can mix the RNA into the formamide sample dye and store for a later gel, you only heat right before running the samples. RNA will inter- and intra-anneal, especially rRNA, so you have to maintain a denaturing environment: high formamide, high urea, and high temperatures.

Once samples are prepared, they can be stored at -80 for many years. If you plan to run the gel soon, you can keep them in the fridge, or on the bench if running right away.

For the gel(s):

The gel is cast in the Bio-Rad mini protein gel system, but any thin vertical system will do - I would practice one first. If it comes out OK, make another and run it.

For the buffer, we use our TBE version at 2X (100 mM Tris, 100 mM borate, 1-2 mM EDTA), there should be a 5X somewhere, if you use the last, make more. See the Moore Lab TBE page.

To make 5 mL of gel mix (for one gel, double for 2), weigh 2.5 g of high quality urea and put it in a 15 mL tube. Add 5X TBE to make ~90-100 mM TB (2X in our case, 2/5 * 5 mL = 2 mL of 5X), and then add acrylamide mix to desired final concentration (maybe 1.25 mL, see next paragraph), that will bring the volume closer to the 5 mL goal, vortex briefly and shake down (remove air from urea), then add water with squirt bottle to make 5 mL. [As an aside, when adding a volume such as 1.25 mL, you can set a 1 mL pipetter for 625 uL and pipette twice, it's easier and more accurate that adding 1 mL, then 250 uL.]

The acrylamide volume you add depends on the mix of the stock and your final goal. We commonly use a stock of 40% 19:1 Acrylamide:Bisacrylamide from National Diagnostics for RNA and DNA gels. Most RNAs resolve well with a 10% gel; that is, 10% final concentration of acrylamide, not 10% of 5 mL volume.

Cap it and votex until urea is dissolved, then a bit more to make sure. Some people microwace the mixture (cap loose) for a few seconds to heat the sample and speed up the urea solvation, but make sure to cool it before proceeding.

It helps to slightly de-gas this sample before polymerizing - you can connect a rubber stopper to a vacuum line and suction it it to the top of the open 15 mL tube, a few bubbles will form right away. If you release the vac, they will go back into solution, you don't want that, you want them out of the solution when you release vac; so, rotate/tap tube to dislodge bubbles and let them fuse then with the open atmosphere above the sample, don't worry about every one, just the bulk of them, then release vac. Doing this will allow better and more thorough polymerization and sharper well edges. It's also fun.

Get the casting stand set up and ready with clean plates (cleaned with soap and water until a slow water-flow travels smoothly over the surface, wiped with some ethanol spray in one direction, and dried). Get a 10-tooth comb ready - note that the comb is not to be jammed all of the way in, there should be little tabs that allow you to set them on the upper edge of the smaller plate, that is plenty, you're only loading 5-15 uL.

When ready (casting stand set up, pipette waiting, maybe a few kimwipes on hand) , add 15 uL of APS (from recently made 10% stock), mix gently, then add 5-10 uL TEMED - once it's added, the gel will start to polymerize - you have to get the gel poured fast - either with repeated 1mL additions, using a 5 mL pipetter, or pouring it in. The amount of TEMED and the dissolved oxygen will affect polymerization rate. If you have more than one to cast, it may be a good idea to cut back to ~5 uL TEMED per gel. The polymerization rate will vary and your experience has to develop. For your first time, you can prepare 10 mLs of gel, add APS, and then make 5 x 1 mL aliquots. Add different amounts of TEMED to each, mix, and monitor the tubes by inverting them periodically. Pick a suitable volume of TEMED that gives you enough time to pour the gel, removed potential air bubbles, and place a comb into the poured gel before polymerizing. Then, use that fraction of TEMED for the remaining 5 mL for the final gel. As you fill the casting mold, bubbles may get trapped; you can pause, tilt and tap the glass on the bubble to dislodge the bubbles. It can be easier to tilt the unit until the bubble contacts the surface of the liquid and pops, then continue filling. Experience will allow you to pour without bubbles every time. Making sure your plates are clean will help too.

Fill the gel plates until the gel solution slightly over-flows the lower plate, then carefully insert comb, without making bubbles under the wells or between them. Inserting the comb will cause additional gel to pour over, that is good. If, during the seating of your comb, you backed out a bit, the gel will be below the lower glass plate, in this case pipette more liquid acrylamide in between the wells to make it overflow again. Leave the excess sitting on the top of the smaller plate - it is in contact with air and won't gel.

Leave the comb in place and don't disturb the unit until the gel is done polymerizing (in good lighting, you will see a refractive index boundary between unpolymerized and polymerized gel near the teeth and in between them. After some time, those boundaries won't move any more. Monitoring the formation of the tooth separations is key; the polymerization boundary will rise from the body of the gel toward the exposed surfaces between the teeth, then slow and stop. Depending on the degassed state, the amount of catalyst, and the percentage/type of acrylamide, that position will vary, but it should be close to the top of the tooth gaps (after all, these define the well depths).

Before pulling the comb, bring the unit to a sink (will be covered with acrylamide and crystallized urea) - rinse briefly with deionized water, not too much, just to get rid of junk. Release the gel frames while rinsing and make sure the surface that will contact the tank seals is free of debris. Carefully and slowly remove the comb: pulling too fast will cause a suction that causes the well separators to release from the plates and shift. While not ideal, they can be coaxed back into position with careful use of a loading tip, just assume there may be sample leakage between those wells. Take gel to running station for set up in a clamp and tank, Leave the casting stand in the sink for now.

Running Gels

Assemble the tank system and fill inner chamber with running buffer (same as in gel, 2X of our TBE) until it fills the upper chamber. Pause and make sure there is not a dramatic leak by monitoring the level in the top tank. It is normal for the level to drop a few millimeters by leaking near the top of the gasket, but it should stop and a decent amount over the lower plate should remain (~0.5-0.75 cm). If it is leaking too much, lift the gel and carefully pour the inner chamber buffer into the tank, release and reset the gels against the gasket, pour buffer in from the tank to fill the inner chamber again, and reset the gels into the tank. Add more buffer to the top tank until it over flows into the lower chamber and sufficiently covers the bottom electrode wire (about where those rounded humps are, or more).

Using a 10 mL syringe and a cut needle (can be left near gels and reused over and over), briefly and gently pulse the insides of each well with running buffer to remove any debris and unpolymerized acrylamide. Don't blast the well separators out of position.

Start running the gel without samples to pre-warm at 300V volts. Make a note of the current, this value can be used to ensure you made the reagents correctly/consistently between runs. Here, you are warming the gel and letting urea leech into the wells, these events are desired. Monitor your gel to make sure the upper chamber is not severely leaking, if it is, release and reset the gels.

Let it run 20-30 mins. Meanwhile, go back to sink and wash your gel casting system - removing acrylamide now is critical, if it hardens and dries it messes up subsequence casting, especially on the foam seals. Set the parts somewhere to dry.

About 5 mins before you plan to load, get your RNA samples out and put them in hot sand, >~85-100 deg, should be near gels.

Without disturbing the gel wells, stop the power, remove the lid. Transfer your RNA samples to a rack (will be hot, can use kim-wipe). If the top buffer level is a bit low, use the well washing syringe to transfer some warm buffer from the bottom tank to the upper reservoir. While the samples and the gel are still hot/warm, you want to load: using a gel loading tip carefully transfer the desired volume into the well, UNDER the urea that leached out. If you cast your wells too deep, your brand of tips may not reach to the bottom. You have to deliver just above the gel in the well. Use two hands with both elbows on the bench. The sample should spread out and form sharp edges and stay covered with urea (may be hard to see, the goal is to reduce exposure of the RNA to regular TBE, it will refold). Avoid blowing bubbles through the sample. Repeat for each sample.

Replace the lid and run at full voltage again for the desired time. These gels can be run short to resolve smaller RNAs or long to separate longer ones, like rRNAs. One trick to resolve both forms with a few samples is to load one side of the gel, run it, then stop and load adjacent gels for the last (short) run. In doing so, you will have the same RNA samples resolved to different extents on the same gel. If you have more samples, you can prepare two gels and load them both. After the shorter run time, remove a gel and replace it with a dummy plate, and continue to run the second. I forget times, but ~70 mins resolves smaller RNAs. The bromophenol blue will move off of the gel on longer runs, just monitor the time.

Staining

After running, disassemble the gel (these gels can be weak and slimy, easy to tear, be careful), drop into a dish with dH2O, rock it a few seconds, then decant water and add stain solution. With SYBR Green II, it takes about 10 minutes and the tray can be covered with aluminum foil to reduce light exposure. After straining, transfer gel to a container with water and move to imager. Place gel flat on saran wrap on UV and image; the filter depends on dye, most machines have a SYBR-green like filter. If the stain is reused, the RNA from previous gels starts to contaminate the stain and the background will go up, but generally a few uses of a batch of stain is OK if it's relatively fresh and kept dark in the fridge.