Northern Blots(Ambros Lab)

by Michael Koelle and Tory Herman, adapted from Sambrook et al., "Molecular Cloning"

We found that both formaldehyde and glyoxal gels work very well for electrophoresis of RNA; we only prefer glyoxal gels because the fumes from the formaldehyde gels are unpleasant. Most protocols suggest using nitrocellulose rather than charged nylon membranes for blotting; the latter are said to give higher background. We have been using uncharged nylon ("Nytran" from Schleicher and Schuell) with good results. Because a good nylon Northern blot can be kept for years and reprobed a dozen or more times with good results, it seems a shame to use nitrocellulose, which is so physically weak that the blot is unlikely to last more than a couple of probings without tearing.

As with all RNA work, precautions against RNAse contamination should be taken. Solutions should be DEPC treated; reagents and plasticware should be from fresh packages set aside for RNA work; glassware, spatulas, stir bars, etc. should be decontaminated by baking overnight at 180deg. C. Because electrophoresis boxes are often used to analyze DNA samples that have been RNase treated, they are of special concern. A special gel box and combs should be set aside for RNA work only. If there is some question that a gel box or comb has been contaminated, they can be cleaned by thorough washing with detergent, rinsing, soaking with 3% hydogen peroxide for 10 minutes at room temperature, and finally rinsing in DEPC treated water.

Solutions:

Caution: Wear gloves and avoid getting DEPC on your skin.

RNase free water (make several liters to make up the running buffer. Having some 100 ml bottles is also good.)

To ddH 2 O, add DEPC to 0.1%

Shake to get the DEPC droplets into solution

Leave at 37deg. overnight

Autoclave 20 minutes to destroy the DEPC

0.1 M sodium phosphate pH 7.0 (make 1 liter)

Adjust to the appropriate pH by mixing ~2/5 volume of monobasic with ~3/5 volume of dibasic 0.1 M sodium phosphate

Make using clean technique, add DEPC to 0.1%

Shake to get the DEPC droplets into solution

Leave at 37deg. overnight

Autoclave 20 minutes to destroy the DEPC

6 M glyoxal (deionized)

Glyoxal is purchased as a 40% (6M) solution (e.g. from Sigma). Glyoxal readily oxidizes in air. Oxidation of the solution can be detected by the lowering of its pH as carboxylic acids accumulate. These charged acids can be removed by passage through a mixed-bed resin (e.g. Bio-Rad AG 501-X8). This must be done quickly, minimizing exposure of the glyoxal to air, or it will just reoxidize as you work!

Our method is to use a baked spatula to load about 5 mls of resin into each of ~3 small Bio-Rad dispo-columns (from a fresh bag). Measure the pH of the starting material by putting a few drops on pH paper; it will probably be <1. Pour about 10 mls of solution over the first column, collecting the eluate in an RNase free 15 mls centrifuge tube. Then pass the eluate over the column again, collecting in a second tube. Continue by passaging the solution twice each over the second and third columns, or until the pH of the solution appears to have reached a steady state. Measure the pH after each column by putting a few drops on pH paper. The goal is to reach ~pH 5, although in practice a steady state may be reached at about pH 4.5. Quickly aliquot the deionized solution into RNase free screw cap tubes (~50ul aliquots), seal the caps tightly, and freeze at -80deg.. Each aliquot should be used only once and then discarded.

Loading buffer: 50% glycerol, 10 mM sodium phosphate pH 7.0

Use glycerol from a fresh unopened bottle, and DEPC treated water and 0.1 M sodium phosphate pH 7.0.