What should be the minimum probe length, the GC content and annealing temperature? Is a DNA or RNA probe more preferable? Is there any software to help design probes or can I just pick up a specific region that essentially binds only with my gene of interest? A radioactive or a fluorescent probe- which is more preferable?
For an mRNA, or longer RNA Northern, the best probe is a radioactive in vitro transcript. RNA-RNA interaction is the strongest. The probe should be more then 100 bp, in that case we don't talk about annealing temperature. You have to optimize you Northern conditions, if the hibridization is not good. Radioactive probes allow you detect very low signals, so you can use stringent conditions during hibridization. The probe should be specific to your target RNA, you can choose the best regions by BLAST. Despite of the best design, it could happen, that your probe won't work well.
Interesting... I performed my last Northern blotting well before the genome sequence was completed and at that time it was all radioactive and we were preferentially taking regions from the 3' ends (far from proteins, because gene families may cause cross hybridization) and checking that there were no repetitive elements in the 3' UTR, because they would cross-hybridize with other expressed repeats.
Now with the genome availability and other transcriptome, it should be much easier. Again, longer is better. No repeat elements, no protein families. Radioactive is better when testing conditions, because one can wash the filter until the signal decreases considerably by stringent washing, in order to identify specifically hybridized RNAs only.
Again with the genome availability I guess one can design probes that work much better than in the past.
I would recommend random prime labeling of a DNA fragment. Pick a fragment from the 3' UTR or some other non-conserved sequence and use the random prime label kit from Roche. Standard hybridization temperatures are 65oC in an aqueous buffer or 42oC in 50% formamide. You do not need to optimize the temperature for each probe. Fragments for labeling should be at least 300 bp The great thing about northerns is that the membrane can be stripped and reprobed multiple times for different genes. Use Gene Screen and cross link the RNA after transfer for a blot that last a long time.
While radioactive RNA probes can potentially improve detection of low abundance transcripts, all hybridization steps have to be in nuclease-free solutions, with extreme care due to the instability/degradation of RNA transcripts. Also, there is the problem of high background. I would say try first as Dr. Gregory Dressler suggests. In our lab, we usually use similar conditions as he described though our probes are a bit longer, about 500bp. We never had to optimize any of the conditions, and we had good results with a variety of probes.
I agree, there is much less problem with DNA probes. And interestingly, I had much less problem in Church's buffer on 68oC then with a formamide buffer on 42oC. But I have never had problem with the stability of the RNA probe. After transcription I used it immediately, and then reused the whole hybridization mix several times in general. Everything depends on the task. Try first the simplest, optimize if you are not satisfied. You are lucky to work on human, in plants everything is about optimization :)
Hi all, I need to do Northern blotting to fish out a microRNA. Can I use the mRNA or cDNA of the protein which is complementary to my microRNA as a probe?
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