Northern Blotting: Efficient RNA Staining and Transfer

(original protocol by R. M. Fourney, S. Miyakoshi, R. S. Day III, and M. C. Peterson (Focus 10:1), modifications of this protocol were carried out by Carol Alosi)

Methods

Glassware should be silanized and baked at 200 ℃for > 4 hours. Plasticware should be dep-treated and autoclaved.

Buffers

All solutions should be dep-treated and autoclaved except SDS and Denharts which should be made with dep-treated, autoclaved H ₂ O. The pH of the 37% formaldehyde solution should be adjusted to 7.0.

10x MOPS/ EDTA Buffer: 0.2 M Mops[3-(N-morpholino) propanesulfonic acid], 50 mM sodium acetate, 10 mM EDTA adjusted to pH 7.0 and autoclaved.

Electrophoresis Sample Buffer (freshly prepared prior to loading or stored at -20oC in small aliquots): 0.75 ml deionized formamide, 0.15 ml 10x MOPS, 0.24 ml formaldehyde, 0.1ml deioinzed RNase-free H ₂ O, 0.1 ml glycerol, 0.08 ml 10% (w/v) bromophenol blue.

Electrophresis buffer: 1x MOPS/EDTA buffer.

Other solutions required: 37% formaldehyde (pH 7.0), 10x SSC, 1.0 mg/ml ethidium bromide in deionized RNase-free H ₂ O.

Sample Preparation

1. Isolate RNA by the method of your choice.
2. Dissolve the sample in 25 mM EDTA, 0.1% SDS (or TE 10/1).
3. Add 1-3 ug poly (A)+ RNA or 10-30 ug total RNA to an RNase-free micro -centrifuge tube.
4. Adjust volume to 5 ul with DEPC-treated, autoclaved water. If necessary, concentrate dilute samples by lyophilization.
5. Add 25 ul Electrophoresis Sample Buffer and 1ul ethidium bromide solution and heat at 65℃ for 15 min and do not put on ice.
6. Load the sample on the gel.

Gel Preparation and Electrophoresis

1. Add 1.0-1.5 g agarose, 10 ml 10x MOPS and 87 ml diethyl pyrocarbonate (DEPC)-treated autoclaved H ₂ O to an RNase-free flask (we prefer the sterile orange cap flasks).
2. Dissolve agarose and let cool to 50℃.
3. In a fume hood, introduce 5.1 ml 37% formaldehyde into the agarose solution, gently mix, and than pour the gel into an RNase-free 11 x 14-cm gel tray.
4. Allow the gel to sit for 1 h before use (if waiting longer than an hour to load gel cover gel in saran wrap).
5. Prior to loading the gel, flush sample wells by pipetting electro- phoresis buffer in and out of the wells.
6. Load wells and electrophorese the gel at 30-60 V (constant voltage) at room temperature for 2-6 h. Bromophenol blue migrates ~10 cm into the gel.