Liu Lab
1.
Prepare all equipment for gel:
 Rinse plates with cold water, then clean with 1% SDS and warm water. Be careful not to scratch plates and that you rinse until all soap is removed.
 Wash spacers, combs and gel tank with warm water. Rinse with distilled water and ethanol, and then again with water.
 If using the BioRad II system, clamp the two plates together on one side and place on the gel-pouring mold.
 Make sure that you have all the buffers and reagents necessary at hand (TBE, SSC, DEPC Water, Formaldehyde gel loading dye,
Acrylamide). See last page for recipes and ordering instructions.
2.
Pouring the gel:
 To make a 15% gel start by measuring out 7.2g of Urea in a
50ml conical blue-capped tube.
 Add 1.5ml of 10xTBE
 Add 5.6ml of 40% Bis-Acrylamide (19:1)
 Place the tube in a 37C heating block and wait until the urea dissolves completely
 Add Nuclease-free water to the 15ml mark of the blue-capped tube
 Filter sterilize the mixture using a filter and syringe. Filter it into a previously baked small beaker.
 Add 75  l of 10% APS (not older than 2 weeks)
 Add 15  l of TEMED
 Swirl the mixture and pour the gel into the mold using a pipette. Be careful not to make bubbles.
 Leave to polymerize about 1 hour
3.
Pre-running the gel:
 Carefully remove comb and place the gel into the buffer reservoir.
 Fill both reservoirs with 1xTBE. Rinse out any extra urea or gel pieces from the wells using a syringe and 1xTBE.
 Pre-run the gel at constant 50mA for 1 hour or until the gel reaches 50C.
4.
Load and run the gel:
 For a marker use two nucleotides, 21 and 24b each. These need to have a complementary antisense probe designed for them.
For example: You can use a miR172 5’ to 3’ oligonucleotides which are 21 and 24 bp long. At the same time, you need to have a complementary antisense probe that will be labeled with radioactive ATP and used to visualize the marker. Mix the marker to a concentration of 50nM.
 Prepare 30  g of total RNA in no more than 15  l of DEPC water.
Mix the RNA with an equal volume of 2x Fomamide dye. For

Liu Lab example, if you are using 10  l of RNA, use the same amount of dye.
 For the markers, mix 1  l of 50nM marker 21bp, 1  l of 50nM marker 24bp, 8  l of DEPC water and 10  l of dye.
 Prepare all the RNA samples as mentioned above.
 Heat samples at 70C for 5 minutes and then immediately transfer tubes on ice.
 Turn off the power on the gel.
 Rinse the wells again using a syringe. Make sure no urea is blocking the wells.
 Carefully load samples in the following order: Marker, empty well, sample 1, sample 2, etc., empty well, and then control samples. The control sample can be 5  g of RNA from one or all samples.
 Load samples.
 Run at a constant 50mA until the BPB dye is at the bottom of the gel.
5.
Transfer gel and generate blot:
 Pre-cut nylon membrane to size of gel with clean gloves and razor
 Pre-cut five pieces of thick Whatman filter paper to the size of the gel
 Wet the semi-dry blotting apparatus (Biorad) with a small volume of 0.5xTBE
 Soak four of the filter paper pieces and the membrane in 0.5xTBE
 Pry apart the glass plates and cut the upper corner of the gel, as well as the wells
 Soak the gel in 0.5XTBE for 5 minutes
 While the gel is soaking, set up the transfer sandwich on the semidry blotting apparatus
 The apparatus should go as follows: two pieces of wet Whatman paper, membrane, gel RNA side up, two pieces of wet Whatman paper. Be sure to roll out all the bubbles after you have set up the transfer sandwich.
 Transfer the miRNA at 4C for 45 minutes.
6.
EDC cross linking of RNA to membrane
 While waiting for the transfer of gel, prepare fresh the EDC crosslink solution:
Add 245  l of 12.5M 1-methylimidazole to 9ml DEPC-treated water. Add 300  l of 1M HCl to make the pH 8.0. Measure out
0.753g EDC and add to the same tube. Add water to 24ml.
 Disassemble, mark blot and rinse in 1xTBE
 While rinsing blot in TBE, soak a piece of thick Whatman paper in the EDC solution.
 Lay blot RNA side up on the EDC soaked Whatman paper, wrap in saron wrap and incubate at 60C for 1 hour.

Liu Lab
 Once incubation is done, you can store blot in between two pieces of thin Whatman paper or proceed to prehybridization.
7.
Pre-hybridization:
 Wet blot in 1xTBE, roll membrane with RNA side in, and insert into hybridization bottle.
 Add ~12.5ml of prehyb/hyb buffer and prehybridize at least for 30 minutes at 37C.
 While blot is prehybridizing, proceed to probe generation
8.
Probe generation:
 Set-up 5’-end labeling of probe: in the following way:
1  l 5  M miR172c Probe
9  l  -32ATP (1.67
 M)
2  l PNK Buffer
7  l Water
1  l PNK Enzyme
20  l Total
 Incubate at 37C for 1 hour
 Complete purification of radiolabeled probe following
Ambion small RNA purification protocol
 Denature probe at 90C for 5 minutes and place on ice
 Add denatured probe the prehybridization/hybridization solution, that is already on the membrane
 Hybridize at 42C overnight
 Remove hyb solution
 Wash blot in tube with ~50ml of pre-warmed wash buffer.
Start with 3x10 minute washes at 42C with 2xSSC, 0.1%SDS
Recipes:
5xTBE
54.0g Tris Base, 27.5 g Boric acid, 20.0ml EDTA (pH 8.0), bring to 1L with autoclaved water
PreHyb/Hyb Solution:
100ml 20xSSC, 8ml 1M Na2HPO4 pH 7.2, 280ml 10% SDS, 12ml 100x Denhardt’s solution. Just before use, denature 100  l of Herring sperm, add it to the prehyb/hyb solution and add solution to membrane.