PAGE separation of small RNAs

For the resolution of RNAs between 15 and 500 nucleotides in length, depending on the percentage acrylamide used.


Formamide buffer (0.01M EDTA, 0.1% bromophenol blue, deionised formamide)

Gel set-up

PAGE small RNAs tableNB: bromophenol blue migration and run time based on an 8×5 cm mini-gel

  1. Put together the running assembly, fill with 0.5x TBE, remove the comb and clean the wells with running buffer
  2. Pre-run the gel for 1 hour at 300 V

Sample preparation & gel running

  1. Resuspend 1 μg RNA in 2.5 μl ddH2O (RNAse-free) and add 7.5 μl formamide buffer
  2. Denature at 65°C for 15 minutes, spin briefly, place on ice; load samples taking care not to overflow into neighbouring wells
  3. Run gel at 300 V or until the dye front runs off the gel

Staining & transfer

  1. Remove the gel from the assembly and place in 0.5x TBE containing 50ng/ml ethidium bromide for 10 minutes; de-stain in 0.5x TBE for 10 minutes
  2. Transfer to positively charged nylon membrane (e.g. Zeta membrane, Bio-Rad) using a semi-dry electroblotter (e.g. transblotter, Bio-Rad)
  3. Pre-soak the membrane and two pieces of extra thick filter paper in 0.5x TBE for 10 minutes
  4. Set-up the transfer stack (bottom: filter paper, membrane, gel, filter paper: top) and transfer for 1 hour at 250 mA
  5. Cross-link in a Stratalinker or similar

Pre-hybridise and hybridise as per standard northern protocol