Barbara Rueger, Julia Thalhammer, Irmgard Obermaier, and Stefanie Gruenewald-Janho

Boehringer Mannheim Customer Service Lab, International Support, Mannheim, Germany

2. MATERIALS AND METHODS

Electrophoresis conditions and sample preparation

See Table I for required solutions. Prepare all solutions with sterile dimethyldicarbonate-treated distilled water, then autoclave; we use dimethyldicarbonate instead of diethylpyrocarbonate because of DMDC's lower toxicity. When preparing Northern blots, use sterile solutions and sterile trays as much as possible. Treat gel boxes with ethanol prior to the run.

1. Prepare a formaldehyde/agarose gel. We use MOPS-formaldehyde (2% final concentration) agarose gels exclusively and typically prepare them with 1-1.5% Agarose MP.

2. Prepare fresh formamide/formaldehyde RNA loading buffer each day. If not prepared fresh for each use, the formaldehyde will react with the formamide during storage. The bromophenol blue will turn from blue to green during storage and degrade the target.

3. Denature the RNA target sample for 10 min. at 65°C in RNA loading buffer, and place on ice immediately. For example, to 5 µl (100 ng, 50 ng, 5 ng) of target RNA, add 10 µl RNA loading buffer.

We do not use glyoxal denaturation of RNA because (i) glyoxal must be freshly deionized prior to use for best results, and (ii) it has been reported that glyoxal may react with RNA to form complexes that are inhibitory to efficient transfer.

4. Load the dry gels, add electrophoresis buffer to the edges of the gel, allow the RNA to move into the gel at high voltage, and then add electrophoresis buffer to submerge the gel.

Note that our RNA loading buffer does not contain Ficoll or glycerol, which are generally used to simplify loading.

5. Run the gel with 1X MOPS buffer at 25 V overnight under a fume hood. Running gels overnight produces better separation. We include 1 mg/ml ethidium bromide in our running buffer to stain the RNA during the separation; ethidium bromide does not interfere with successful Northern blot hybridizations as has been occasionally reported. However, when running gels at high speed for a short time, we have sometimes observed that one half of the blot stains black after detection. This is probably a result of ethidium bromide running in the opposite direction of the RNA. In a short run, ethidium bromide does not pass through the total gel, and the "running front" becomes visible on the blot.

RNA transfer

1. Pre-equilibrate the gel in sterile 20X SSC for 10 min. This removes any remaining formaldehyde.

2. Transfer the RNA to a nylon membrane, using sterile 20X SSC as transfer buffer. To obtain the highest possible sensitivity, a positively charged nylon membrane is required. We always use the positively charged nylon membrane from Boehringer Mannheim, which has been specially developed to give the highest sensitivity with the DIG System. It is tested for a lower and an upper limit of charge, which is extremely important when working with nonradioactive probes at the high probe concentrations required for optimal results. Each lot is also quality controlled with DIG-labeled probes in a dot blot detection. Uncharged membranes can be used if highest sensitivity is not required.

3. Fix the nucleic acids to the membrane. In our labs, we have almost exclusively switched to UV-cross linking (at 120 mJ) because of the speed and convenience (i.e., with the Stratalinker auto-program). However, baking for 30 min at 120°C has proved equally efficient with the Boehringer Mannheim nylon membrane. When baking the membrane, wash it in 2X SSC or sterile water to remove the 20X SSC prior to baking.

4. After UV cross-linking, briefly wash the membrane in water, and air dry it. This removes residual 20X SSC, which is essential prior to hybridization. Alternatively, you may briefly wash the membrane in 2X SSC or sterile distilled water immediately after transfer (i.e., prior to cross linking); however, this produces bands that are not as sharp.

Probe labeling

The ß-actin RNA is available already DIG-labeled (Cat. No. 1 498 045). For the CTF1 probe, we used the labeling procedure summarized here:

1. Linearize 1 µg template DNA with a restriction enzyme that generates 5' overhanging ends (1).

2. Treat linearized template DNA with phenol (2), ethanol precipitate.

3. Perform in vitro transcription with T7 RNA polymerase according to the pack insert of the DIG RNA Labeling Kit.

4. Evaluate the efficiency of labeling reaction by a standard direct detection as described in The DIG System User's Guide for Filter Hybridization (3).

We recommend a final labeled probe concentration of 100 ng/ml hybridization solution.

Prehybridization, hybridization, and stringency washes

1. In a sealed plastic bag, prehybridize the blot in 25 ml hybridization solution (lacking the probe) for 30 min at 68°C. We use DIG Easy Hyb buffer for all blot applications. This buffer has been specially developed for nonradioactive probes and has many advantages. It is quality controlled in a Southern blot and a Northern blot hybridization and has been evaluated for all blot applications. It contains urea instead of formamide, so it is not toxic. Hybridization conditions corresponding to the presence of 50% formamide are applied.

The hybridization temperature of 68°C has been empirically identified to be optimal for most RNA:RNA hybrids in the presence of 50% formamide. In rare cases when the prospective hybrid will have very high G+C content, this temperature may be increased.

2. Denature the probe for 5 min at 100°C, then immediately chill on ice.

3. Prewarm 3 ml (calculated for 67 cm²) hybridization buffer to 68°C, then add the probe to a concentration of 100 ng/ml.

4. Hybridize the blot at 68°C overnight in a sealed plastic bag.

5. Remove the blots carefully from the bag. Immediately wash them twice, 5 min per wash, with shaking in low stringency wash buffer 1 (2X SSC, 0.1% SDS) at room temperature.

During the washes, shake the tray, and use excess amounts of buffer to prevent the membranes from sticking to the tray and partially drying. Never allow the blots to dry between prehybridization and the final washes.

6. Wash the blots twice, 15 min per wash, with shaking in prewarmed high stringency wash buffer 2 (0. lX SSC, 0.1% SDS) at 68°C.

It is extremely important to prewarm the washing buffer for these stringency washes.

After the stringency wash, the blot may be stored dry at 4°C (if the blot will not be stripped and reprobed) or placed in wash buffer for immediate immunological detection.

Chemiluminescent detection

1. Centrifuge the vial of antibody conjugate in a microcentrifuge for 5-10 min to pellet precipitates that form during storage. Use antibody conjugate from the surface of the solution only.

If not removed, the precipitates will lead to dark spots on the membrane during chemiluminescent detection. The antibody can be stored again at 4°C, but this centrifugation procedure must be repeated prior to the next use.

2. Perform chemiluminescent detection according to the pack insert, or apply the "Transparency technique". See Table 2 for a summary of both methods of applying the chemiluminescent substrate. We used the following "Transparency Technique" (steps 3-5 below) to apply the chemiluminescent substrate in our examples.

3. Place the membrane on a transparency. Add approximately 500 µl of diluted chemiluminescent substrate per 100 cm² membrane.

4. Cover the membrane with a second transparency sheet, and incubate for 5 min at room temperature.

5. Allow excess liquid to drip off, and seal the "sandwich."

6. If CSPD® chemiluminescent substrate was used, preincubate the membrane for 10 min at 37°C.

This activates the decay of the unstable intermediate formed upon turnover of the substrate by alkaline phosphatase. If CDP~Star^TM substrate was used, omit this step.

7. Place the "sandwich" in an X-ray cassette, and expose to X-ray film.

When using the CSPD substrate, exposures may also be performed at 37°C to obtain a faster result.