MicroRNA Northern Protocol
Reminder ­ Wear Gloves, lab coat and eye protection!!!
Gel Setup Using Protean II system
- Clean materials with 10% SDS and rinse thoroughly with ddH20
- Set up gel apparatus as per normal (use thick spacers) 1.5mM
- Make up 15% Denaturing Gel
# of Gels
5x TBE Buffer
- Allow gel to polymerize for 1 hour
- Assemble gel apparatus and add the running buffer (0.5X TBE)
- Clean out wells with running buffer ­ make sure there are NO leaks!!!
- Pre-run the gel at 180 volts for 30 min
- Rinse wells right before loading sample
RNA Prep and Gel Running
- You want to load 20ug of total RNA per lane ­ add DEPC H2O up to 20uL
- Add 20uL of formamide to your RNA sample
- Heat RNA at 65ºC for 10 min
- Chill on ice for 1 min
- Add some Bromophenol Blue loading dye
- Load samples and run at 180 volts until the dye reaches the bottom of the gel
- After running gel, stain with EtBr in 0.5X TBE for 5 min. Destain in 05X TBE. This will allow you to visualize tRNAs and 5S RNA for normalization. Place a ruler down as a reference.
- Rinse gel in 0.5X TBE to remove excess EtBr.
Gel Transfer with Trans-blot Semi-Dry transfer cell
- Use the GeneScreen Plus membrane and cut to size slightly larger than the gel.
- Soak membrane in dH20 for a few seconds
- Soak membrane in transfer buffer (0.5X TBE) for 15 minutes
- Soak 2 pieces of whatman paper in 0.5X TBE
- Set up transfer as such ­ From Bottom (anode) to top : whatman, membrane, gel, whatman, cathode plate. Make sure to roll out any bubbles
- Transfer at 400mAmps for 1 hour. The voltage will start out low but increase by the end of the transfer
- Wash blot in 0.5X TBE to remove any traces of the gel
- Place wet membrane on a wet sheet of filter paper and UV Crosslink at optimal setting
- Store membrane at 4ºC until use
To a screw top tube, add this
2ul 10x PNK Buffer
2ul Oligo Probe
1ul T4 PNK
5ul 32P gATP
Mix and incubate at 37 degrees for 45 min
Add 80ul of TE
Run through G-25 column
- Prehyb membrane in Ultrahyb Oligo solution for 0.5 hours at 42º C. Add 1mL/10cm2 of membrane.
- Add your 32P-labeled probe to the prehyb solution and incubate 12-24 hours at 42íC
- Pour off hyb solution and wash membrane as follows ­ 2 washes for 30 min. in 2xSSC/0.5% SDS
- Cover membrane in saranwrap
- Place in phosphor-imager cassette for ~4 hours.
Boil membrane in stripping solution (0.1X SSC, 1% SDS) for 10-30 min