Blotting

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The principle of transferring a nucleic acid from a gel to a membrane, commonly known as blotting, is identical whether DNA or RNA is used. Here we describe a standard method for upward capillary blotting (15) of RNA to a nitrocellulose filter.

3.2.1. Preparation

1. During the blotting process the RNA is susceptible to degradation until the RNA has been fixed to the nitrocellulose (in Subheading 3.2.3., step 5). Therefore, make sure glass trays, glass plates, supports, and a glass test tube are RNAse-free (see also Note 1). Wear gloves.

2. After electrophoresis, transfer the gels to a glass tray and soak in 10X SSC with gentle shaking for at least 1 h, replacing the 10X SSC buffer twice. Increasing the time for this step to several hours seems to improve the overall quality of the blot.

While the gels are soaking, do the groundwork for setting up two Northern blots (see Subheading 3.2.2.).

3. Cut 16 pieces of Whatman paper of gel size (100 x 125 mm).

4. Cut two pieces of Nitropure membrane (Micron Separation) of 105 x 130 mm, i.e., slightly larger than the gel.

5. Cut four pieces of Whatman paper the size of the support tray.

Fig. 1. Upward capillary transfer of RNA. The 10X SSC blotting buffer is drawn through the paper wick, the RNA agarose gel, and the nitrocellulose membrane into the stack of filter paper and paper towels, transferring the RNA transcripts out of the gel onto the nitrocellulose membrane.

6. Cut or fold a large amount of paper towels to gel size, enough to make two stacks of approx 10 cm.

Steps 1-9 describe setting up one Northern blot. This will have to be done for both gels as prepared and described in Subheading 3.1.

1. Pour approx 0.5 L of 10X SSC in a glass tray and place a glass plate on supports (see Note 9) in the middle of the tray.

2. Pre-wet the nitrocellulose membrane in MilliQ H2O for 5 min and then in 2X SSC for another 5 min.

3. Wet the two sheets of tray size-Whatman paper throughout in 10X SSC and drape over the support in such a way that the Whatman filter paper is overhanging like a wick into the buffer on all four sides. Remove any air bubbles between the filter paper and the support by rolling a glass 150-mm test tube (or an RNase-free solid glass rod or glass pipet) over the wet paper.

4. Using the test tube pour approx 5 to 10 mL of 10X SSC to flood the Whatman paper and then place the gel on the filter paper on the support, taking care not to trap any air bubbles under the gel. Gently roll and press the gel with the test tube to ensure that no air bubbles are trapped.

5. Flood the gel with a little 10X SSC and carefully place the pre-wetted nitrocellulose onto the gel, taking care not to trap any air bubbles. Try to put the membrane straight down in one go. Gently roll the test tube over the membrane to ensure good contact and to remove all air bubbles.

6. Wet thoroughly a stack of four pieces of gel-size Whatman paper in 10X SSC. Flood the nitrocellulose with a little 10X SSC and place the sheets of wet filter paper on top of the membrane. Again, roll the test tube over the paper to remove any air bubbles.

7. Add another approx 0.5 L of 10X SSC to the glass tray to raise the level to just under the top of the support.

8. Place strips of Parafilm tightly against the gel on all four sides on the overhanging tray size-Whatman paper. This step prevents filter paper and tissues short-circuiting the upward capillary flow through the gel and membrane.

9. Place another four pieces of dry gel-size Whatman paper on top of the wet filter paper, followed by a stack of paper towels, a glass plate, and a bottle filled with 300 to 400 mL of liquid as a weight.

3.2.3. RNA Transfer and Immobilization

1. Leave blot setup overnight as described above, allowing 12 to 20 h for RNA to be completely transferred (see Note 10). Make sure the stack of paper towels will not tilt, which will ensure even transfer across the membrane.

2. Dismantle, date, and label each blot with a unique identifier, and mark with a pencil the position of the wells. Also pencil-mark the side of the membrane that has faced the gel (RNA-side).

3. Wet the membrane in 3X SSC and briefly and gently "wash" the membrane— i.e., carefully remove any pieces of agarose that are stuck to the membrane by means of stroking with a gloved finger.

4. Place the membrane on a clean piece of Whatman paper and air-dry at room temperature.

5. UV-crosslink the RNA to the dry membrane (RNA-side up) with a dose of 120 mJ.

6. Take a picture of each blot (RNA-side down) using a UV transilluminator to check RNA integrity and blotting efficiency. Exposure of blots to UV light should be minimized.

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