Practical Tips for Optimal Northern Analysis

Northern assays require RNA (total- or poly(A)- selected) to be resolved on a agarose gel under denaturing conditions, transferred to a membrane and immobilized for subsequent hybridization. Various inconsistencies in the procedure, particularly the transfer set-up, can greatly effect the assay's outcome. Here, we describe several technical points which can make any Northern analysis a success.

Gel Parameters - Less is More

• Pour gels as thin as possible. The gel must be thick enough to provide well depth to accommodate sample volume, but the thinner the gel, the faster and more efficient the transfer will progress.
• Transfer is also impeded by agarose gel concentrations greater than 1.2%.

Gel Transfer to Membrane

Transfer of RNA to a membrane (Northern blotting) can be accomplished using capillary transfer or electroblotting. Electroblotting utilizes a manufactured apparatus and is extremely efficient as long as the manufacturer's instructions are followed. Capillary transfer utilizes a stack of dry paper towels to draw the transfer solution though a wick of blotting paper (such as Whatman 3MM), the gel, the membrane and into the dry towels. The capillary transfer set up can be upward or downward (Figure 1). Regardless of the method chosen there are hints that can optimize any capillary transfer.

Standard Upward Capillary Transfer

NorthernMax Downward Capillary Transfer

Figure 1. NorthernMaxà Downward Capillary Transfer Versus Standard Upward Capillary Transfer.

• Measure twice, cut once - Capillary transfer requires not only a membrane cut to the size of the gel, but also several sheets of blotting paper and a stack of paper towels several centimeters in height. To conserve transfer solution and materials, it is useful to cut any excess gel away from your samples and measure the gel prior to cutting blot materials. A standard office paper cutter aids this process considerably. The stack of paper towels, which should be three or more centimeters high, is easily cut using a sharp razor blade or utility knife and a straight edge.
• Hands off - When cutting the membrane, it is useful to keep the protective paper on either side of the membrane while cutting and handling. Otherwise, the membrane should be handled only by its edges and with forceps. Fingerprints or gloveprints can contribute to background signal seen on autorads, this is especially true for membranes subjected to nonisotopic detection.
• Get oriented - It is useful to mark the membrane for orientation prior to wetting; e.g. cut off a corner or write the date on the upper right corner. The date can be lightly marked with a pencil on the back of the membrane (opposite side from the RNA).
• Tiny bubbles - When setting up the transfer take care not to introduce bubbles in any of the layer; it is especially important to avoid bubbles between the gel and membrane. As each component is added to the blot, a pipette can be used to gently roll out any bubbles.
• Dodging the issue - It is important that the transfer not be "short-circuited". This occurs when any of the blotting materials come into contact with the transfer solution wick. For efficient transfer, the solution must only pass through the gel and membrane with no route around the two. To avoid "short circuiting", cut all materials to the size of the gel and mask around the gel with strips of parafilm or plastic wrap.
• Bottoms up (or down, as the case may be) - Be sure the side of the gel corresponding to the bottom of the wells is against the membrane. This minimizes the distance the RNA must migrate out of gel to the membrane.

Cross Linking

After transfer is completed the membrane should be treated to immobilize the nucleic acid, otherwise there is a loss in signal intensity (Figure 2). There are several ways in which this is accomplished, including baking in a vacuum or conventional oven (30 minutes to 2 hours at 80°C), UV crosslinking or microwaving. At Ambion, we find UV crosslinking to be the most convenient and effective method.