Matthew Freeman

NORTHERN BLOTS USING RIBOPROBES


The advantage of using riboprobes; for northerns is that you can make very hot probes, and the extra stability of RNA:RNA hybrids may increase the sensitivity. They are therefore useful if you are looking for similar, but not identical, transcripts, or if your transcript's abundance is very low. There are significant disadvantages also. In order to make the hottest possible probe, a relatively large amount of radioactivity is used, and also there can be a problem with spurious bands (for example I often see the same band when I have used Bluescript vectors; as the probe, presumably because there is a degree of similarity between some vector sequences and some Drosophila transcript). The latter problem is usually solved by increasing the stringency of the final washes: the different signals melt off very efficiently at different temperatures, and you could normally expect that the most stable hybrid represents the 'real' transcript. Nevertheless, this could clearly cause problems in some situations, as well as actually being an advantage in others (such as deciding which of several transcripts was most similar to a given probe). It should be noted that these probes are RNA, and are therefore sensitive to RNAses, which contaminate most glassware, and all hands. Use normal precautions for working with RNA and all will be well. The protocol below takes a very cautious approach; in practice you can usually be more sloppy.

Labelling the Probe:

The following recipe gives the hottest probe that can be made from available nucleotides. It uses 750µCi to make a single probe, and therefore should only be used in extreme situations. You can reduce the amount used by using ribonucleotides of lower specific activity and/or reducing the reaction volume; the critical factor is that the concentration of the hot nucleotide is at least 8µM. The whole procedure is based on the one in the instructions that come with the Stratagene Transcription Kit;; there is a discussion of all the stages in that booklet.
Northern blots;
1) Dry down 750µl of 3000Ci/mmol, 1mCi/ml rATP in the Speedvac (I split it into two tubes, thereby halving the time taken).

2) Resuspend the hot nucleotides in the following reaction:
5µl 5X Transcription Buffer* (Stratagene)
1µl 10mM rCTP
1µl 10mM rGTP
1µl 10mM rUTP
1µl 0.75M DTT
1µl RNAse Block (25U, Stratagene; RNAsin is OK too)
1µg DNAÝ
10U T3 or T7 Polymerase
Water to 20µl

*5X - 200mM TrisHCl, pH8, 40mM MgCl2, 10mM spermidine, 250mM NaCl.
Ý The DNA is Bluescript cut down stream of the insert, treated with 200µg/ml proteinase K, 15 minutes, 37°, phenol extracted, ethanol precipitated, and resuspended in DEPC-treated water.

3) Incubate at 37° for 30 minutes.

4) Add 100µg RNAse-free tRNA, an equal volume of 4M ammonium acetate, and 2.5 volumes ot ethanol. Precipitate. By measuring the radioactivity in the pellet and the supernatant, it is possible to get a very approximate idea of the incorporation; it should be greater than 50%. Resuspend in boiling salmon sperm DNA just before setting up the hybridisation.

Northern Blot

The northern is run under standard conditions.

Hybridisation
1.Prehybridise for at least 4 hours at 65° in:
50% formamide
5X SSC
1X PE*
150µg/ml salmon sperm DNA (phenol extracted)

*5X PE; is: 250mM Tris-HCl, pH7.5
0.5M sodium pyrophosphate
5% SDS
1% polyvinyl pyrolidine
1% ficoll
25mM EDTA
Make in DEPC-treated water, heat to 65° to dissolve. Cool to about 37°, and add 5% BSA (fraction 5) to 1% final. Heat again to 65° for 15 minutes. Store indefinitely at room temperature.

2) Hybridise in the same mix with the probe (I resuspend the probe in a little just-boiled salmon sperm DNA). Hybridise at 65°, overnight or longer.

3) Wash in 0.1X SSC, 0.1% SDS at 65°. This is rather low stringency (see above), and any signal you see should be checked by rewashing the filter (which should not be allowed to dry completely) at increasing temperatures. For example I have found three bands on a northern that only gives one with DNA probes. One of the spurious bands comes off between 65° and 70°, and the other by 75°. In this case the real band still hybridises up to 85°.

Using this protocol, a transcript that is normally visible in about 12 hours (using oligo-labelled probes), is visible in 1 - 2 minutes, suggesting that these really are very sensitive northerns.