MicroRNA Northern Protocol

 

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
Northern Gel
# of Gels
 
 
40% Acrylamide
 18.75 mL
 
5x TBE Buffer
 5 mL
 
dH20
 10 mL
 
Urea
 21 g
 
10% APS
 400 ul
 
TEMED
 40 ul
 

 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
 

Post 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
 

Labelling Probes

               To a screw top tube, add this

                              10.4ul dH20

                              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

               Count probe

 

Probing Membranes

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 42?C
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.
 

Stripping Membranes

               Boil membrane in stripping solution (0.1X SSC, 1% SDS) for 10-30 min