Membrane Transfer and Crosslinking for RNA

Analysis of mRNA expression in tissue or cell culture is often done by Northern blot or ribonuclease protection assay (RPA). Northern assays require the total RNA to be resolved on a denaturing agarose gel first, then transferred to a membrane and immobilized for subsequent hybridization. Nonisotopic RPAs, which utilize probes labeled with modified nucleotides (e.g. biotin, digoxigenin, fluorescein, or suitable hapten), are transferred to a membrane from denaturing polyacrylamide gels for detection by a secondary detection scheme (e.g. streptavidin/avidin conjugates, or anti-digoxigenin and anti-fluorescein antibodies).

Since different types of gels are used for these techniques, the mode of transfer is different in each case. Agarose gels are used for Northerns because of their wide range of resolving power and large loading capacity. Their porosity allows efficient passive transfer of nucleic acids onto a membrane. Polyacrylamide gels characteristically have much sharper resolution, but lower loading capacities. The nature of polyacrylamide gel matrices does not permit efficient transfer by passive diffusion, thus an electroblotting method is used instead.

Choice of Membrane

There are several types of commercially available membranes suitable for RNA analysis, composed of different materials and carrying different charges. The common ones are made of nylon and nitrocellulose, and may be neutral, negatively or positively charged. Nylon (polyamide) membranes are made of the most durable material, but can shrink or warp if exposed to organic solvents. Nitrocellulose tends to tear easily in washing steps and becomes very fragile and brittle if baked. It is also incompatible with secondary detection steps, since protein easily absorbs to the surface and does not specifically bind to the hapten on the probe. The surface of neutral membranes actually comprises equal amounts of positive and negatively charged molecules. The overall net charge is zero, but spotty background can result due to areas of with higher densities of positive charges. Negative membranes give the cleanest background, but result in poor specific signal. Positively charged membranes give the best signal of all, but they also result in higher background. Although the signal-to-noise ratio is lower on positively charged membranes than on other membrane types, the lower level detection limit they permit offsets that disadvantage. For this reason, we recommend using Ambion's BrightStar™-Plus membranes (Cat #10100), which are positively charged nylon and have a high affinity for nucleic acids.

Optimal Transfer Conditions

Denaturing Agarose Blotting
The best low-tech method for agarose transfer is a passive, slightly alkaline, downward elution. This procedure, in comparison to upward transfer, is much faster, and therefore results in tighter bands and more signal.

The composition of the transfer buffer is usually a 5X SSC/10 mM NaOH solution. These mildly alkaline conditions shear the RNA into smaller fragments and denature it as it is deposited onto the membrane. A brief protocol for assembly (see Figure 1) and transfer is as follows:

1. Remove top end of gel by slicing through wells with a razor blade. For orientation purposes, cut a small notch in the upper right hand corner of the gel.
   
   
2. Cut paper towels and filter paper to roughly the same dimensions of the gel. The top sheet of filter paper is wetted with transfer buffer before the membrane is placed on the stack.
   
   
3. Wet the membrane and lay it on top of the paper stack. Notch the membrane in the upper right hand corner. Use a glass pipet to roll out any air bubbles between the membrane and the filter paper (nucleic acids do not transfer through air).
   
   
4. Wet the bottom of the gel and lay on the membrane, aligning the notches. Again, smooth out any bubbles between the gel and membrane.
   
   
5. Wet another filter paper piece and lay on top of the gel, smoothing out bubbles. Lay a few more pieces of filter paper on top of the stack.
   
   
6. Use a few lengths of 3MM Whatman chromatography paper to create a bridge from the buffer tank to the paper stack.
   
   
7. Cover the stack with the gel casting tray to keep the stack wet. Do not add weight to compress the stack.
   
   
8. Check to be sure the buffer bridge is not touching the paper towels below the gel, and that all buffer transfer is only possible through the gel.
   
   
9. Continue transfer for 1-2 hours. Do not attempt to check progress, or else the alignment may be disturbed.
   
   
10. Disassemble and proceed with the crosslinking method of choice, discussed later.

Polyacrylamide and Agarose Gel Blotting
Transfer from polyacrylamide gels requires more force than is offered by passive elution. The highly crosslinked matrix does not allow passive transfer in efficient, quantitative, or reproducible yield. Thus, polyacrylamide gels should be transferred by electroblotting. This method has shown that a ³²P/biotinylated RNA probe is transferred at 100% efficiency to Ambion's BrightStar-Plus membranes, with no material left behind in the gel and none passing through the membrane:

The protocol is simply that of the manufacturer's recommendations for their apparatus. The method described here has been developed with the Owl transfer blotter:

1. Cut six pieces of filter paper to the size of the gel to be transferred.
   
   
2. Prepare 100 ml of 0.5X TBE electrophoresis buffer for wetting the papers.
   
   
3. Wet two of the filter papers and place them on the cathode plate of the electroblotter. Use a glass pipet to roll out any air bubbles that may inhibit transfer.
   
   
4. After gel electrophoresis, separate the glass plates and immobilize the gel onto a piece of filter paper. Lay the gel/filter paper on top of the wetted papers, gel side up.
   
   
5. Cut the upper right-hand corner of the membrane for orientation purposes, wet the membrane, and place on top of the gel. Smooth out any air bubbles with a glass pipet.
   
   
6. Wet the last three filter papers and place on the stack. Take care to squeeze out any trapped air.
   
   
7. Wet the general area of the anode plate that will be in contact with the paper stack.
   
   
8. Place the anode plate on top and secure firmly, but not so tight that contacts will occur outside of the stack.
   
   

9. Electrophorese for 30 min. at 200 mA (constant current setting).
   
   
10. Disassemble and proceed with the crosslinking method of choice, discussed below.

Crosslinking Methods

There are two common methods for immobilizing RNA on a membrane; both work equally well. These two options are given based on the availability of equipment in your lab. UV crosslinking is one of the most popular methods, using either a hand-held UV lamp at short wavelength, or a commercial crosslinking device. The other common method baking the membrane in an oven at 80°C.

UV Crosslinking
Shortwave UV light causes the nitrogenous bases in RNA, mostly uracil, to become highly reactive and to form covalent linkages to amine groups on the surface of the membrane. Damp membranes require an exposure of approximately 120 millijoules/cm². This is usually equivalent to the "auto-crosslink" feature on commercially available, calibrated UV crosslinkers. If a calibrated instrument is not available, it is possible to use standard laboratory equipment such as transilluminators and handheld ultraviolet lamps to fix RNA targets to a membrane. Care must be taken not to under or overexpose the RNA to UV light — both of which will decrease hybridization signals. Usually a one minute exposure with 254 nm light or three minutes with 302 nm light is sufficient. To ensure maximum sensitivity, however, the following experiment should be carried out.

1. Prepare a gel with five identical lanes containing 1 µg of RNA each. Run the gel and transfer it to the membrane.
   
   
2. Carefully cut the membrane into identical strips containing one lane each. Wrap these individually in a single layer of UV-transparent plastic wrap.
   
   
3. Put the strips face down on a transilluminator (or face up if using a handheld light source). Be sure to wear UV-opaque eyewear.
   
   
4. Expose individual strips to UV light for 30 seconds, 45 seconds, 1 minute, 2 minutes, and 5 minutes. Be sure the strips are treated exactly as they will be during actual use, especially the degree to which they are allowed to dry before irradiation.
   
   
5. Probe the blot for an abundantly to moderately abundantly expressed message, according to your Northern blot protocol.
   
   
6. The strip having the highest intensity signal corresponds to the optimal exposure time for a particular membrane with a particular UV source. This experiment should be repeated occasionally, as the energy output of a particular device may change over time.

Baking
Baking works by heating the membrane to drive out all water solubilizing the RNA. A large component of RNA is its hydrophobic nucleotide bases, which make hydrophobic contacts with aromatic groups on the membrane. This interaction is affected by heating in an oven at 80°C for 15 min. The only danger in baking is that the membrane can be damaged if the heat is not regulated to prevent temperatures from rising much higher than 100°C.

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