- 15% denaturing acrylamide gel [1X TBE, 7 M urea, 15% acrylamide (19:1 acryl:bis-acryl)] Since this gel is used to separate the ~21-mer mature miRNA species from RNA that is significantly longer, the resolution does not need to be optimal. We recommend using a 0.75 mm spacer, and a comb appropriate for the sample volume.
for 15ml | Component |
---|
6.3 g | Urea (high quality, e.g. Ambion cat #9900) |
1.5 ml | 10X TBE |
5.6 ml | 40% Acrylamide (19 acryl:1 bis-acryl, e.g. Ambion cat #9022) |
to 15 ml | distilled deionized water |
Stir at room temperature until the urea is completely dissolved, then add:
75 μl | 10% ammonium persulfate |
15 μl | TEMED |
Mix briefly after adding the last two ingredients, which will catalyze polymerization, then pour the gel into a
prepared mold immediately.
- 10 X TBE: Tris-Borate-EDTA Buffer; Ambion Cat #9862–9864
- Ambion Gel Loading Buffer II for denaturing PAGE (Cat #8546G, 8547)
- Linear Acrylamide: Ambion Cat #9520
- 1M NaCl: Dilute Ambion’s 5 M NaCl Cat #9759 with high quality nuclease-free water to 1 M.
- 100% ethanol: ACS grade or higher quality
- 80% ethanol: Make 80% ethanol from high quality nuclease-free water and ACS grade (or better) 100% ethanol.
- Ambion MEGAclear™ Kit Cat #1908
Important
Before starting the miRNA clean-up, preheat the Elution Solution (from the MEGAclear Kit) to 95°C.
- Add the following to each miRNA sample, and pass the mixture through a MEGAclear Filter Cartridge
- Place a MEGAclear Filter Cartridge in a 5 ml syringe barrel, and mount the assembly on a vacuum manifold.
- Add linear acrylamide and ethanol to each sample, vortexing after each addition to mix thoroughly:
Amount | Component |
---|
1.5 μl | Linear Acrylamide (5 mg/ml) |
to 60% | 100% ethanol (typically 18 ml) |
- Pipet the RNA mixture from the previous step into the MEGAclear Filter Cartridge. Vacuum pressure will pull the solution through the filer.
- Wash with 4 ml of 80% ethanol
With the vacuum still on, slowly pipet 4 ml of 80% ethanol into the column, allowing the vacuum to pull the solution through the filter.
- Using a centrifuge, wash with 500 μl of 80% ethanol, then dry the filter
- Place the MEGAclear Filter Cartridge in a Collection Tube, and pipet 500 μl of 80% ethanol into the column.
- Centrifuge for 1 min at 5,000 x g and empty the Collection Tube.
- Centrifuge again at 10,000 x g to remove excess ethanol.
- Move the MEGAclear Filter Cartridge to a fresh Collection Tube.
- Elute the miRNA in 2 x 50 μl 95°C Elution Solution and dry the miRNA to completion
- Apply 50 μl heated (95°C) Elution Solution (from the MEGAclear Kit) to the MEGAclear Filter Cartridge, and leave at room temp for 2 min.
- Centrifuge for 1 min at 10,000 x g. Purified miRNA will be centrifuged into the Collection Tube.
- Repeat the elution with a second 50 μl aliquot of heated Elution Solution, centrifuging purified miRNA into the same tube.
- If you isolated miRNA from more total RNA than is needed for a single mir Vana miRNA Labeling Kit reaction, split the eluted miRNA into single reaction-sized aliquots. (Use miRNA obtained from 5–50 μg of total RNA per mir Vana miRNA Labeling Kit reaction.)
- Place miRNA sample in a vacuum concentrator and dry completely.