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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.
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, which are positively charged nylon and have a high affinity for nucleic acids.
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:
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:
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
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.
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.