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MOLECULAR BIOLOGY: WORKING WITH DNA
NUCLEIC ACID PROBES
NORTHERN BLOT HYBRIDIZATION AND WASH USING RIBONUCLEIC ACID PROBES (RIBOPROBES)
Northern Blot Hybridization and Wash Using Ribonucleic Acid Probes (Riboprobes)
The advantage of using ribonucleic acid probes (riboprobes) for Northerns is that you can make very radioactive probes, and the extra stability of RNA:RNA hybrids may increase the sensitivity. Riboprobes are therefore useful if you are looking for similar, but not identical, transcripts or if your transcript's abundance is very low. There are disadvantages of using riboprobes as well. In order to make the most radiolabeled probe possible, a relatively large amount of radioactivity is used. In addition, there can also be problems with spurious bands. This problem is usually solved by increasing the stringency of the final washes. Different background signals will melt off very efficiency at different temperatures, and you could normally expect that the most stable hybrid represents the "real" transcript. These probes are RNA, and are therefore sensitive to RNase's, which contaminate most glassware and all hands. Use normal precautions for working with RNA and all will be well. The protocol takes a cautious approach. Procedure
A. RNA Transcription
1. Vacuum centrifuge to dry down 750 μl of 3,000 Ci/mmole (1 mCi/ml) rATP. (CAUTION See Hint #4)
2. Resuspend the radionucleotide in the following reaction (see Hint #1):
5 μl 5X Transcription Buffer
1 μl 10 mM rCTP
1 μl 10 mM rGTP
1 μl 10 mM rUTP
1 μl 0.75 M DTT
1 μl RNase block (25 Units final concentration)
1 μg DNA (see Hint #2)
10 Units T3 or T7 RNA Polymerase
Add distilled deionized water (ddH2O) to final volume of 20 μl
3. Incubate the solution for 30 min at 37°C.
4. Add 100 μg RNase-free tRNA.
5. Add an equal volume of 4 M Ammonium Acetate and 2.5 volumes of 100% Ethanol.
6. Mix well by inversion, microcentrifuge to pellet the RNA and discard the supernatant (see Hint #3).
7. Invert tubes over a paper towel and allow pellet to air dry (1 to 2 min).
8. Resuspend RNA in boiling salmon sperm DNA "JUST BEFORE" setting up the hybridization or resuspend RNA in ddH2O.
B. Northern Blot
1. Pre-hybridize the filter for at least 4 hours at 65°C in Hybridization Buffer.
2. Hybridize in hybridization buffer at 65°C overnight (or longer, determine empirically).
3. Remove the filter and wash in 0.1X SSC/SDS at 65°C (this is low stringency, to increase the stringency you can increase the washing temperature, for other wash strategies see alternative Northern Protocols).
4. Using this protocol, transcripts that are normally visible on X-ray film with the use of intensifying screen after 12 hours are visible in 1 to 2 minutes (although the exposure time needs to be determined empirically).
0.1X SSC/SDS 0.1% (w/v) SDS
Hybridization Buffer Prepared in DEPC-treated ddH2O (see Hint #5)
50% (v/v) Formamide (CAUTION See Hint #4)
150 μg/ml Salmon Sperm DNA (phenol extract before use)
5X SSC Solution
1X PE Solution
0.75 M DTT PE Solution (5X) Prepared in DEPC-treated ddH2O (see Hint #5)
Heat the solution to 65°C to dissolve. Cool to approximately 37°C and add 5% BSA to a final concentration of 1%. Heat again to 65°C for 15 min then cool to room temperature.
5% (w/v) SDS
0.5 M Sodium Pyrophosphate
1% Polyvinyl Pyrolidine
250 mM Tris-HCl, pH 7.5
1% (v/v) Ficoll
25 mM EDTA
10 mM rUTP 5% BSA 5% (w/v) Bovine Serum Albumin, Fraction V
Prepared in DEPC-treatedH2O (see Hint #5)
10 mM rGTP SSC (20X) Prepared in DEPC-treated ddH2O (see Hint #5)
3 M NaCl
0.3 M Sodium Citrate
10 mM rCTP 4 M Ammonium Acetate 5X Transcription Buffer (Stratagene) 40 mM MgCl2
200 mM Tris-HCl, pH 8.0
10 mM Spermidine
250 mM NaCl
DEPC Autoclave 20 min to inactivate DEPC
Also see Hint #5
Shake vigorously and let stand overnight.
0.1% (v/v) DEPC to ddH2O
RNase Block 25 Units/μl RNase Block BioReagents and Chemicals
RNA Polymerase, T3 or T7
DNA, Salmon Sperm
Bovine Serum Albumin (BSA), Fraction V
Diethyl Pyrocarbonate (DEPC)
1. The recipe gives the most radioactive probe that can be made from available nucleotides. It uses 750 μCi to make a single probe, and therefore should only be used in extreme situations. You can reduce the amount of radionucleotides (or use one with a lower specific activity). The critical factor is that the concentration of the radionucleotide is at least 8 uM.
2. The protocol has been successful using BlueScript vector cut down stream of the insert. The solution is then treated with 200 μg/ml Proteinase K for 15 min at 37°C. Protein is extracted with a standard Phenol extraction and Ethanol precipitation. The washed pellet is resuspended in DEPC-treated water.
3. By measuring the amount of radioactivity remaining in the supernatant it is possible to get a very approximate idea of the incorporation (incorporation should be greater than 50%).
4. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
5. To minimize degradation of RNA by RNases, wear gloves when handling samples and reagents and change gloves regularly while working. Treat water and solutions with DEPC (Diethyl Pyrocarbonate) to inactive RNases and use solutions prepared with RNase-free water and equipment. For more information about precautions when working with RNA, see Reference Pages under Working with RNA.