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