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

1. Gel electrophoresis

1. Prepare 15% acrylamide/8 M urea gel solution.
   CAUTION: Acrylamide must be handled with care, because it is a neurotoxin. Always wear gloves, a lab coat, and goggles when handling it.
   1. In a 150 mL glass beaker, combine 22.5 mL of premixed 40% acrylamide/bis-acrylamide 29:1 solution, 6 mL of 10x TBE, 28.8 g of urea, and RNase-free water to a total volume of 59 mL. Gently stir the solution.
      NOTE: Depending on the RNA substrate length, altering the percentage of polyacrylamide may improve the resolution between unphosphorylated and phosphorylated RNA.
   2. To dissolve the urea, heat the solution in the microwave for 20 s, stir the liquid, and immediately place the solution back into the microwave for another 20 s. Gently stir the solution until the urea completely dissolves.
   3. Slowly cool the solution by placing the glass beaker into a shallow water bath containing cold water. Make sure that the level of cold water surrounding the glass beaker is above the level of solution inside the glass beaker. This will promote efficient heat transfer. Wait 5 min.
      NOTE: Do not continue with this protocol if the glass beaker feels warm; the water should be below 25 °C. If it still feels warm after 5 min, then replace the water in the water bath with fresh cold water and wait another 5 min.
   4. Filter and degas the solution using a 0.22 μm disposable filtration unit to remove particulates and microscopic air bubbles.
2. Pour the denaturing gel
   1. Use soap and warm water to clean a short and long glass plate designed for gels with overall dimensions of 31.0 cm x 38.5 cm. Spray each glass plate with 95% ethanol and wipe the glass to remove any moisture.
      NOTE: One of the two plates can be siliconized to prevent damage to the gel when separating the glass plate sandwich. However, this step is not necessary because this protocol is designed to visualize the RNA without separating the glass plates.
   2. Place the long glass plate horizontally on top of a box so it is elevated off the benchtop.
      CAUTION: Acrylamide is toxic, therefore the gel pouring station must be covered in bench paper that can absorb any spilled liquid and be immediately placed in a waste bag after the procedure is complete.
   3. Place a clean 0.4 mm spacer along the long edges of the long glass plate.
   4. Lay the short glass plate atop the long plate and ensure the edges of the short plate, long plate, and spacers are aligned. Clamp each side using three evenly spaced metal clamps.
   5. Add 24 μL of TEMED to the 15% acrylamide/8 M urea solution prepared in step 3.1 and mix the solution.
   6. Add 600 μL of 10% (w/v) APS to the solution in step 1.2.5 and gently mix the solution.
   7. Immediately pour the solution between the glass plates.
      NOTE: To avoid bubbles, tap the glass as the solution is poured. 
   8. Carefully add a clean, 0.4 mm, 32 well comb to the top of the glass plate sandwich.
   9. Allow a minimum of 30 min for the acrylamide to polymerize.
3. Run the denaturing gel
   1. Set the heat block to 75 °C.
   2. Remove the metal clamps holding the glass plate sandwich together and thoroughly wash and dry the glass plate sandwich.
   3. Place the glass plate sandwich into the gel apparatus with the short plate facing inward.
   4. Prepare 0.5x TBE running buffer by combining 100 mL of 10x TBE with 1.9 L RNase-free water. Add 600 mL of the running buffer to the upper and lower chambers of the gel apparatus.
   5. Gently remove the comb and rinse the wells thoroughly using a syringe. NOTE: This step is critical for removing urea from the wells.
   6. Pre-run the gel for 30 min at 50 W. Voltage is approximately 2,000 V. CAUTION: This gel apparatus operates at a high wattage and users should exhibit precaution.
   7. Rinse the wells thoroughly using a syringe.
      NOTE: This step is critical for even loading of sample into the wells.
   8. Pulse spin the quenched reactions. Then incubate the tubes at 75 °C for 3 min. Repeat the pulse spin.
   9. Immediately load 10 μL of sample per well and run the gel for 3 h at 50 W.
      NOTE: Fluorescently labeled RNA is light sensitive; therefore, cover the gel apparatus with foil.
4. Image the denaturing gel.
   1. Turn off the power supply and drain the upper chamber of the gel apparatus.
   2. Wash and dry the outer side of the glass plate sandwich using soap and water. Cover the glass plate sandwich with foil while transporting the gel.
   3. Mount the glass plate sandwich onto the stage of a laser scanner capable of quantitative and sensitive fluorescence detection.
      NOTE: This gel is extremely thin for maximum resolution. For this reason, this protocol is designed to avoid the difficulties of removing the gel from the glass plate sandwich by directly visualizing the fluorescently-labeled RNA through the glass plates. The use of commercially available low-fluorescence glass plates will increase the captured signal by improving the signal-to-noise ratio.
   4. Set the excitation and emission wavelengths of the laser scanner for the desired fluorophore.
      NOTE: The optimal excitation and emission wavelengths may differ for specific fluorophores. For the FAM fluorophore, the excitation and emission wavelengths are 495 nm and 535 nm, respectively.
   5. Define the area to be visualized on the laser scanner stage and image the gel according to the manufacturer's instructions (Figure 1).