MicroRNA Northern Protocol
Reminder ­ Wear Gloves, lab coat and eye protection!!!
Gel Setup Using Protean II system
materials with 10% SDS and rinse thoroughly with ddH20
- Set up
gel apparatus as per normal (use thick spacers) 1.5mM
up 15% Denaturing Gel
# of Gels
5x TBE Buffer
- Allow gel to polymerize for 1 hour
gel apparatus and add the running buffer (0.5X TBE)
out wells with running buffer ­ make sure there are NO leaks!!!
the gel at 180 volts for 30 min
wells right before loading sample
RNA Prep and Gel Running
want to load 20ug of total RNA per lane ­ add DEPC H2O up to 20uL
20uL of formamide to your RNA sample
RNA at 65ºC for 10 min
on ice for 1 min
some Bromophenol Blue loading dye
samples and run at 180 volts until the dye reaches the bottom of the gel
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.
gel in 0.5X TBE to remove excess EtBr.
Gel Transfer with Trans-blot Semi-Dry transfer cell
the GeneScreen Plus membrane and cut to size slightly larger than the gel.
membrane in dH20 for a few seconds
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
at 400mAmps for 1 hour. The
voltage will start out low but increase by the end of the transfer
blot in 0.5X TBE to remove any traces of the gel
wet membrane on a wet sheet of filter paper and UV Crosslink at optimal
membrane at 4ºC until use
a screw top tube, add this
10x PNK Buffer
5ul 32P gATP
and incubate at 37 degrees for 45 min
80ul of TE
through G-25 column
membrane in Ultrahyb Oligo solution for 0.5 hours at 42º C. Add 1mL/10cm2 of
your 32P-labeled probe to the prehyb solution and incubate 12-24 hours at
off hyb solution and wash membrane as follows ­ 2 washes for 30 min. in
membrane in saranwrap
in phosphor-imager cassette for ~4 hours.
membrane in stripping solution (0.1X SSC, 1% SDS) for 10-30 min