TITLE: Denaturing RNA electrophoresis in urea polyacrylamide gels (Urea-PAGE) and semi-dry Northern Blot transfer
AUTHORS: Alice Pawlowski

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">RNA is denatured in 8 M Urea and separated in a polyacrylamide gel electrophoresis (PAGE) based on its molecular weight. Using this method, fragments  between 2 to 500 nucleotids can be separated. The migration of the RNA depends on the concentration of PAA:  the higher the PAA percentage, the better the resolution of  low molecular weight fragments and vice versa. We used 10 % PAA to separate fragments between 50 - 200 nucleotides in 10 ml mini-gels (10.5 x 10 cm) using the Hoefer SE260 Mighty Small system. Subsequently, RNA is transfered from the gel to a nylon membrane (Northern blotting) by using aTrans-Blot SD Semi-Dry Electrophoretic Transfer Cell (BioRad). </div></div></div>

[STEPS]
?. [preparation of Urea-PAA gel]
assemble the gel sandwich stack (see pdf in materials section); align plate bottoms with the spacers on a flat surface before sliding the sandwich in the casting clamp assembly and tithening the screws  (finger tight to prevent breaking of the plates)place the sandwich into the caster, notched alumina plate facing to the back block; check that black silicone  rubber gasket is clean two gels can be prepared in the caster; I suggest always to prepare two (in case that one sandwich is leaky); polymerized gel sandwiches can be stored for later use (see below)prepare the gel according to the table below; 10% PAA is fine to separate fragments of 50 - 200 nt wear Vinyl gloves and goggles! weight 5 g urea and transfer to a 15 ml falcon tubeadd 2 ml 40% PAA and 1 ml 10 x TBEadd a. dest. to a final volume of 10 mlheat at 37 °C with agitation until urea is completely solved (shake occasionaly by hand); this will take a while, you can use higher temperatures (e.g. 55 °C) to speed up the processlet the solution cool downadd 12 µl TEMED and mix by inverting the tubeadd 80 µl APS and mix by inverting the tubepour the gel quickly but carefully between the plates using a plastic pasteur pipetteremove air bubbles by shortly lifting and pushing the caster to the tableinsert the comb immediatelywait until gel gets solid (at least 1h)remove the gel from the caster and rinse the sandwich with destilled water to remove buffer and gel remnantsthoroughly wash caster components in mild detergent and rinse with destilled water immediately after useproceed to step 2 or store the sandwich - wrapped in wet towel paper and foil - for later use at 4-8°C   ABCDEFGHI1% PAA4%5%6%8%10%12%15%20%240% AA/Bis (19:1)1.0 ml1.25 ml1.5 ml2.0 ml2.5 ml3.0 ml3.74 ml5.0 ml3Urea5g410 x TBE1 ml510% APS80 µl6TEMED12 µl7a. dest.to 10 mlweight 5 g urea and transfer to a 15 ml falcon tubeadd 2 ml 40% PAA and 1 ml 10 x TBEadd a. dest. to a final volume of 10 mlheat at 37 °C with agitation until urea is completely solved (shake occasionaly by hand); this will take a while, you can use higher temperatures (e.g. 55 °C) to speed up the processlet the solution cool downadd 12 µl TEMED and mix by inverting the tubeadd 80 µl APS and mix by inverting the tubepour the gel quickly but carefully between the plates using a plastic pasteur pipetteremove air bubbles by shortly lifting and pushing the caster to the tableinsert the comb immediatelywait until gel gets solid (at least 1h)remove the gel from the caster and rinse the sandwich with destilled water to remove buffer and gel remnantsthoroughly wash caster components in mild detergent and rinse with destilled water immediately after useproceed to step 2 or store the sandwich - wrapped in wet towel paper and foil - for later use at 4-8°C
ABCDEFGHI1% PAA4%5%6%8%10%12%15%20%240% AA/Bis (19:1)1.0 ml1.25 ml1.5 ml2.0 ml2.5 ml3.0 ml3.74 ml5.0 ml3Urea5g410 x TBE1 ml510% APS80 µl6TEMED12 µl7a. dest.to 10 ml
?. [run Urea-PAGE]
always wear gloves during all steps!thaw your total RNA samples and marker (RiboRuler Low Range RNA Ladder) on icemix 5 µl total RNA (conc. 5 µg, dilute with RNase free water if needed) with 5 µl 2 x loading dye and incubate 5 min at 95°C (danatures RNA)marker: mix 3 µl LR-ladder with 2 µl RNase free water and 5 µl 2x loading dye and incubate at 70°C for 10 minsnap cool on icemeanwhile assemble the electrophoresis system by inserting the upper buffer chamber in the lower buffer chamber; fix the sandwich to the upper chamber with the clamps, glass plate to the frontoptionally: put a well locating decal to the glass plate to mark the wellscarefully remove the combpour 1 x TBE buffer into the electrode chambersrinse the wells thoroughly with buffer by using a syringe (Hamilton), rinse again directly prior to sample applicationquickly spin down your samples in a microcentrifuge and carefully pipette samples into the wellsclose the safty lid and plug electrodes into a power supplyrun at constant current of 10 mA for 1/3 of the gel length, then increase to 20 mA for the rest of the runstop the run when the bromphenol blue dye (migrates at 12 nt in 10% Urea-PAGE) has just run out off the gel
?. [visualization of RNA]
3+6remove the lid and pour off the bufferremove the clamps and disassemble the gel sandwich: remove the spacers and carefully lift up the glass plate by using one of the spacers as a leveruse the spacer to cut off the wells and the upper right corner of the gel (to mark the orientation of the gel)carefully transfer the gel to a clean tray and shortly rinse the gel in 0.5 x TBEincubate the gel 15 min in 25 ml 0.5 x TBE + 1 µg/ml Ethidiumbromid with gentle agitation in the dark (cover tray with aluminium foil)meanwhile wash the electrophoresis equipment in detergent, followed by dest watercollect the staining solution for re-usage and rinse the gel in 0.5 x TBERNA is visualized in a transilluminator (Gel Doc Imaging System, BioRad), image area  13.4 x 10.0 cm, program nucleic acid- ethidiumbromidesave a gel picture and put the gel back in 0.5 x TBE buffer, proceed to step 4
?. [Northern Blot]
the electrophoretic transfer of the RNA to a nylon membrane is set up in a Trans-Blot SD Semi-Dry Electrophoretic Transfer cell (BioRad)cut a piece of nylon membrane (Hybond N+) and 8 pieces of 3MM Whatman paper in 1 x gel size, soak in 0.5 x TBE bufferhumidify both electrode plates with 0.5 x TBEAssembly of the blotting sandwich (bottom up):4 layers of 3MM papers (1x gel size)nylon membrane (1x gel size)PAA-gel (remove bubbles with a serological pipette)4 layers of 3MM papers (1x gel size)close the lid and plug electrodes into a power supplyrun at constant current of 2 mA/1 cm2 gel for 45 min (= 180 mA for a 10.0 x 9.0 cm mini-gel)you can blot several sandwiches in one transferafter the transfer has finished remove the Whatmann paper and cut the membrane with a scissor along the edges of the gel to have exactly the same size as the gel (including cut off of upper right corner)optional: transfer the gel to 0.5 x TBE and check the transfer in the transilluminator - transfer was successful when you see fluorescence only in the uppermost part of the gel (high molecular weight fragments)transfer the membrane to sheet of towel paper; don't touch the membrane, use foreceps or grap it carefully at the edgesput the membrane on the transilluminator table, RNA site downmake a picture: approx. 0.1 s exposure to UV light (program nucleic acid - ethdiumbromide), image area same as for PAA-gel (10.0 x 13.4 cm); this is necessary for the accurate assignment of signals after hybridization with specific RNA probes!subsequently, cross-link the RNA to the membrane by exposure to UV-light for 120 sec (don't forget!)dry the membrane completely, mark the blot with a pencil and store it between the sheets of Whatman paper or proceed to hybridization with a specific probeAssembly of the blotting sandwich (bottom up):4 layers of 3MM papers (1x gel size)nylon membrane (1x gel size)PAA-gel (remove bubbles with a serological pipette)4 layers of 3MM papers (1x gel size)close the lid and plug electrodes into a power supplyrun at constant current of 2 mA/1 cm2 gel for 45 min (= 180 mA for a 10.0 x 9.0 cm mini-gel)you can blot several sandwiches in one transferafter the transfer has finished remove the Whatmann paper and cut the membrane with a scissor along the edges of the gel to have exactly the same size as the gel (including cut off of upper right corner)optional: transfer the gel to 0.5 x TBE and check the transfer in the transilluminator - transfer was successful when you see fluorescence only in the uppermost part of the gel (high molecular weight fragments)transfer the membrane to sheet of towel paper; don't touch the membrane, use foreceps or grap it carefully at the edgesput the membrane on the transilluminator table, RNA site downmake a picture: approx. 0.1 s exposure to UV light (program nucleic acid - ethdiumbromide), image area same as for PAA-gel (10.0 x 13.4 cm); this is necessary for the accurate assignment of signals after hybridization with specific RNA probes!subsequently, cross-link the RNA to the membrane by exposure to UV-light for 120 sec (don't forget!)dry the membrane completely, mark the blot with a pencil and store it between the sheets of Whatman paper or proceed to hybridization with a specific probeclose the lid and plug electrodes into a power supplyrun at constant current of 2 mA/1 cm2 gel for 45 min (= 180 mA for a 10.0 x 9.0 cm mini-gel)you can blot several sandwiches in one transferafter the transfer has finished remove the Whatmann paper and cut the membrane with a scissor along the edges of the gel to have exactly the same size as the gel (including cut off of upper right corner)optional: transfer the gel to 0.5 x TBE and check the transfer in the transilluminator - transfer was successful when you see fluorescence only in the uppermost part of the gel (high molecular weight fragments)transfer the membrane to sheet of towel paper; don't touch the membrane, use foreceps or grap it carefully at the edgesput the membrane on the transilluminator table, RNA site downmake a picture: approx. 0.1 s exposure to UV light (program nucleic acid - ethdiumbromide), image area same as for PAA-gel (10.0 x 13.4 cm); this is necessary for the accurate assignment of signals after hybridization with specific RNA probes!subsequently, cross-link the RNA to the membrane by exposure to UV-light for 120 sec (don't forget!)dry the membrane completely, mark the blot with a pencil and store it between the sheets of Whatman paper or proceed to hybridization with a specific probe