TITLE: Denaturing PAGE for resolving RNA
AUTHORS: Diep Ganguly, Anna Behle

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol denaturing PAGE separation and identification of total RNA extracts using Urea-PAGE with the BioRad Mini-PROTEAN II / Tetra Cell electrophoresis system. Modified from </span><a href="https://dx.doi.org/10.17504/protocols.io.gszbwf6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">dx.doi.org/10.17504/protocols.io.gszbwf6</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Gel preparation]
Use the following table to prepare the required gel recipe using either 30 % or  40 % PAA (~4 mL=1x0.75 mm gel).  ABCDE1PAA percentage
8 %
10 %
12 %
15 %
230 % / 40 % PAA2 / 2.67 mL2.5 / 3.33 mL3 / 4 mL3.75 / 5 mL38 M Urea5 g5 g5 g5 g410x TBE
1 mL
1 mL
1 mL
1 mL
510 % APS80 µL
80 µL
80 µL
80 µL
6TEMED
10 µL
10 µL
10 µL
10 µL
7DEPC-treated H2Oad 10 mL
ad 10 mL
ad 10 mL
ad 10 mL
ABCDE1PAA percentage
8 %
10 %
12 %
15 %
230 % / 40 % PAA2 / 2.67 mL2.5 / 3.33 mL3 / 4 mL3.75 / 5 mL38 M Urea5 g5 g5 g5 g410x TBE
1 mL
1 mL
1 mL
1 mL
510 % APS80 µL
80 µL
80 µL
80 µL
6TEMED
10 µL
10 µL
10 µL
10 µL
7DEPC-treated H2Oad 10 mL
ad 10 mL
ad 10 mL
ad 10 mL
?. [Gel preparation]
Dissolve urea in 10x TBE, PAA, and 1/2 volume DEPC-treated H2O. Warming solution  (e.g. 37˚C water bath) with gentle agitation will help to dissolve urea.
Wear vinyl or nitrile gloves and work in fumehood when handling PAA (neurotoxic!)
?. [Gel preparation]
Add 80 µL APS (10 %) and 10 µL TEMED to the gel solution and gently swirl once. Immediately pipette solution into gel apparatus ( ~4.5 mL, fill until just before it overflows to prevent bubbles when inserting comb). Retain some gel mixture to check for complete polymerisation.
?. [RNA preparation]
Add required RNA to equal volume 2x gel loading buffer II (Ambion). Up to to 50% can be RNA sample although less is better. Final volumes for loading may vary, however, less than 5 µL results in poor banding when using the Mini protean system (10 µL / gel works well). Keep prepared samples @ 4 °C until ready for denaturation and loading.
RNA loading dye contains formamide. Wear goggles/lab coat/ gloves!
?. [RNA loading]
Once remaining gel solution has polymerised, remove from casting bracket and insert into running bracket (keep combs). Fill interior with 1x TBE and ensure there are no leaks (~5 mins). If leaking, re-adjust and check again. Place bracket into gel tank and fill with 1x TBE.
?. [Visualization]
After sufficient running time switch off power supply, pour out buffer, disassemble gel and clean components. Transfer gel to clean container and rinse once with deionized water.
?. [Gel preparation]
Before preparing the gel, clean all components, gel tank, and surfances with 70 % ethanol (can use 0.1% SDS or RNase Zap to be extra thorough, we do this periodically).
?. [Gel preparation]
Assemble gel plates in the green brackets and place on casting unit. Ensure glass plates are not cracked and are sitting flush against the separator plate and on the rubber mats (to prevent gel leakage).
?. [Gel preparation]
Immediately insert comb tilted at an angle (to minimise trapping air bubbles). Make sure you are wearing appropriate gloves. Lower completely and allow gel to polymerise for >15 mins. Gels can be stored at 4˚C wrapped in plastic (use with 3-5 days).
?. [RNA loading]
Close the lid and plug electrodes into power supply. Separate RNA @ 10 mA / gel for ~1-2 hours (make sure you have a reliable power supply that holds current, particularly as urea has poor conductivity). Allowing the upper marker, from the Ambion loading buffer II, to run off the gel is a reasonable indictor for running time.
?. [Visualization]
Incubate gel in 0.5 µg ethidium bromide / mL 1x TBE for ~15 minutes (or use SYBR gold/green safe stains). Rinse 3x with water and allow to destain for ~2 minutes. Visualize.
?. [RNA preparation]
Determine desired loading amount of RNA per well (e.g. 0.5 - 2  µg RNA). Normalize RNA concentrations accordingly (e.g. 100 - 200 ng/µL).
?. [RNA loading]
Remove combs and rinse individual wells thoroughly.
?. [RNA loading]
During the pre-run, denature RNA samples and ladder, in gel loading buffer II, at 72˚C for 5 min. Once finished, immediately keep at 4˚C until ready to load (minimise time kept at 4˚C).
?. [RNA loading]
Connect electrodes, and pre-run gel @ 10 mA / gel for ~20-30 minutes to remove residual urea.
?. [RNA loading]
Turn power supply off and rinse the well a second time.
?. [Gel preparation]
Once dissolved, allow to cool to room temperature and add DEPC-treated  H2O up to 10 mL.
?. [RNA loading]
Pipette denatured samples into wells slowly while ensuring there are no air bubbles trapped in the pipette tip (Ambion recommends loading while samples are still hot).