Northern blot protocol for small RNAs by PAGE - (Mar/19/2006 )
Does anyone have a good and in depth protocol for northern blotting small RNAs by PAGE? I would like to use a 6% PA 7M urea gel, transfer to nylon membranes, and probe with either labeled oligos or PCR generated probes. The problem is that I don't know any of the details on how to go about doing this. Thank you.
here is protocol for miRNA detection
if you want detect siRNA, probe and buffer might be different for best results.
Total RNA (10 to 50 µg) was separated on 12% polyacrylamide/8M urea gel (Amersham Pharmacia, Uppsala, Sweden) in a Protean II apparatus (BioRad, Hercules, CA) (gel preheated to 55C by electric current and water bath circulation before loading sample) . 21nt RNAs are near the middle position between dye of bromophenol blue and xylene FF in such PAGE gel. Separated RNA in gel was electro-blotted onto Hybond-N+ membrane (Amersham) by Trans-Blot SD semi-dry electrophoretic transfer cell (BioRad). After UV cross-linking and air drying, blotted membrane was prehybridized with Ultrahyb-oligo hybridization buffer (Ambion) at 37~42 ºC for 60 min, hybridized with 32P-end-labeled antisense probe of the target miRNA prepared by T4 polynucleic kinase (Fisher Scientific, Fairlawn, NJ ), and incubated at 37~42 ºC for 3 to 4 hours or overnight. The membrane was washed 2~4 times at 40 ºC with 2x SSC and 0.5% SDS for 15 min and exposed to an X-ray film (Kodak, Rochester, NY) at –80 ºC for signal visualization. To reuse, the membrane was stripped by boiling in 0.1% SDS, cooled to room temperature, and washed once with 2x SSC.