Hi there,

There must be non specific hybridization in your experiment which can be due to different things (too much probe, low stringency, probe cleaved into pieces...).

Hey,

I am not too sure actually. I am used to doing Southerns but not Northerns. So I followed the same protocol as Southern (sans gel treatments) in terms of probe preparation, stringency etc. Plus, there was no background noise whatsoever.

How did you prepare your probe?. How sure that probe is specific to the gene you intended?.

I usually precipitate it after labeling and it gives me perfect southerns. The probe I used in this case is almost 1 kb long (and obviously i had checked the elute on gel before proceeding) and sequence vise it bears no homology with any other gene.

then how did u developed your film (old fashion development by using dark room and ...) or phospho-imaging method?.  in the old fashion simply light -penetration causes darkness of the film and some may interpret it as the effect of probe!. That would have been good if you could simply show the comparative pictures of your southern and northern. otherwise its really hard to write all the possibilities that might have caused this problem. I would simply re-do the northern and check the identity of my probe before everything. good luck Ami

Hi Ami,

You may have already sorted this out, but if not I can hopefully give you some guidance. Since you have experience making probes, it is probably fine, this issue is likely that your wash conditions are not stringent enough, so your probe is sticking non specifically to your RNA and is not being washed off. I will attach our protocol here...it is very simple, and works very well for us. The beginning of the protocol deals with the alkaline transfer method, but starting from step 10 on deals with the handling of the blot for the Northern. The solutions are simple to make and can be stored at RT for a long time.

Hope this helps a bit

Ron 

Hi Bahram,

I prefer to use the conventional method of using an X-ray film for viewing blots. Yeah, I'll repeat it nevertheless. But I'm still confident about my probe. Let's see what happens when I repeat it.

Hi Ron,

No, I haven't sorted out the problem yet. Radioactivity would come next month now, so i have plenty of time to try and improve my protocol. I don't use phosphate buffers, which I think, works well at 65. Mine works best at 42 but your protocol says that non-specific binding increases at lower temperatures so that could be one of the reasons. May be I can try both types of buffers next time. Thanks a lot for sharing the protocol!

Hi Ami,

Just keep in mind that RNA:DNA hybridizations are much more stable then DNA:DNA hybridization, so the conditions that you are used to using for Southern blotting will not work for Northern blotting. I am not sure about your buffer make-up, but the high amount of SDS and the high temperature in the buffers in the protocol I provided are specifically designed to help prevent the nonspecific hybridizations that you are getting.

Good Luck, but I suspect it will just work next time you try. Bummer that your isotope will take so long...that would drive me nuts! This protocol is derived from an even older protocol that we used...I notice it says we make our probes by nick translation, however now we just used a random prime kit (not that it matters).

Cheers

Ron