Northern Blotting：有效的RNA染色和转移

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Northern Blotting技术专题

(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 H2O. 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 H2O, 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 H2O.

Sample Preparation

Isolate RNA by the method of your choice.

Dissolve the sample in 25 mM EDTA , 0.1% SDS (or TE 10/1).

Add 1-3 ug poly (A)+ RNA or 10-30 ug total RNA to an RNase-free micro -centrifuge tube.

Adjust volume to 5 ul with DEPC-treated, autoclaved water. If necessary, concentrate dilute samples by lyophilization.

Add 25 ul Electrophoresis Sample Buffer and 1ul ethidium bromide solution and heat at 65℃ for 15 min and do not put on ice.

Load the sample on the gel.

Gel Preparation and Electrophoresis

Add 1.0-1.5 g agarose, 10 ml 10x MOPS and 87 ml diethyl pyrocarbonate (DEPC)-treated autoclaved H2O to an RNase-free flask (we prefer the sterile orange cap flasks).

Dissolve agarose and let cool to 50℃.

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.

Allow the gel to sit for 1 h before use (if waiting longer than an hour to load gel cover gel in saran wrap).

Prior to loading the gel, flush sample wells by pipetting electro- phoresis buffer in and out of the wells.

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.

Transfer Preparation

Prepare the gel for transfer by soaking it for two 20-minute periods in 10x SSPE at room temperature with gentle shaking.

During the gel washing procedure, prewet the membrane in distilled water for 5 minutes followed by a 5-minute soak in 10x SSPE.

Transfer the RNA in 10x SSPE by capillary action using a sponge to enhance capillary action (we found that not using a sponge worked just as well).

Fix the RNA to the membrane by baking for 2 h at 80℃ or by placing the nylon membrane, with the RNA-side down, on a short wave (254 nm or 302 nm) transillumina tor for 5 minutes.

Comments

If a thick gel or a high concentration of agarose (>1.3%) has been used, it is beneficial to allow the gel to soak for 10-20 minutes in 0.05 M NaOH made up in 1x SSPE. In this latter case, the two 10x SSPE washes are carried out after the NaOH wash. This mild NaOH treatment increases transfer efficiency without greatly degrading the RNA.

The authors recommend using a charge-modified nylon membrane because it has a higher binding capacity than nitroCell ulose and will withstand multiple stripping and reprobing. We found that Hybond N+ work really well.

The RNA bound to the membrane can be viewed for transfer efficiency or photographed under UV transillumination: no staining is required.

Hybridization and Autoradiography

Prehybridize overnight and hybridize for 12-36 h at 42℃with gentle shaking.

Wash the membranes: two 20-minute washes in 2x SSC, 1% SDS at room temperature followed by two 20-minute washes in 1x SSC, 0.5% SDS at 50-55oC.

Autoradiograph with Kodak XAR film at -70℃ for 2-3 days using Dupont Cronex Lightning Plus screens.

Comments

We routinely use the following prehybridization and hybridization solution: 50% deionized formamide, 0.47x Denharts solution, 4.7x SSPE, 0.1% SDS, 0.18 mg/ml denatured salmon sperm carrier DNA, and 5% dextran sulfate. Note that the addition of fat-free mild powder to the prehybridization solution (0.34% final concentration) will decrease the background on membranes which have higher binding capacities.

We use DNA probes labeled to high specific activity (>1x108cpm/ug) using [a-32P]CTP by the random primer labeling procedure (1).

(1) Feinberg, A.P. and Vogelstein, B. 1983. Anal. Biochem. 132, 6.