Probe size for miRNA Northern blot? - (Apr/23/2009 )

I'm tryting to determine if my gene is silenced using small RNAs. I'm planning to do a Northern blot (I've already run and transferred the gel). I have no idea if there is any miRNA, so I'm going to have to scan throughout the entire length of the gene but I don't know the size of the probes I should use. I'd really appreciate if anyone could help with that.
Thanks a lot
Irina

Sorry, I don't really understand your question.
You want to determine the knockdown efficiency or you want to detect the miRNA?
You knock down gene (mRNA) by shRNA or miRNA?
Do you think your gene of interest is silenced by a unknown miRNA?

Functional Screens on Apr 26 2009, 01:44 AM said:

My gene of interest may be silenced by miRNA. I'd like to test that possibility. I've run a 15%PAA 7M urea gel, and the RNA looked good. I transferred it and I'd like to now do a Northern blot, but I'm still working on deciding a good size for the probes. Obviously, I have no idea where such and RNA would be "located" (with respect to the full length mRNA).
Thanks
irina

It is likely that the miRNA is not encoded on the gene that it regulates. What you should be looking for on your mRNA is a miRNA response element (MRE) which, if present, is likely to be in the 3'-UTR. An MRE can have little sequence complementarity with the miRNA (however, the MRE must be mostly complementary to ~8 bases in the seed region of the miRNA).

There are some programs you can use to look for potential MREs in your mRNA of interest, but these are not extremely reliable and should be confirmed by experiment. If you have a potential MRE located, you can try to deactivate the MRE and see if expression is upregulated in the presence of an miRNA when compared to expression from the mRNA containing the putative functional MRE.

Irina on Apr 27 2009, 06:05 AM said:

Do you know which miRNA? If so, why not try reporter assay with cloning different pieces of your gene of interest into the 3' end of reporter to make sure that the presence of miRNA reduces the reporter activity. It has been shown that miRNA regulates gene expression via binding to promoter, mRNA degradation, and translation suppression. It will be hard to look at if you don't know which miRNA potentially silences your G.O.I.

Are you working on plants?

Jon> I'll look into your suggestions and try to find some programs. I'm very new to the whole miRNA issue, and I'm apparently taking the hardest road to learn it :-)
Functional Screens> I don't even know if there is a miRNA. It's just a possiblity I'm trying to test. That's why I'm having so much trouble finding the right approach to figure out if there is one. If you have a better suggestion, I'd be more than happy to take it :-)
andrea> I'm working on a pathogen of the soybean.
Thanks for all the help.
Irina

If you think that the small RNAs you're looking for are highly complementary to your gene makes sense to try a northern. You can use RNA or DNA probes ranging from a few hundreds nt to 1 kb or more. RNA probes are more sensitive, and sometimes they are hydrolyzed by alkali treatment before use, although probably this is not really necessary. There are plenty of methods for small RNA detection by northern without knowing their precise sequence, especially in plant science papers published between 1999 (siRNA discovery) and 2002 (miRNA discovery in plants).
Supposing that a small RNA may guide cleavage of your transcript you can try another strategy. First make a 5' race to map the cleavege site (design primers at the 3' of the transcript). If you find the cleavage site you can design an oligo probe spanning it, that is on the hypothetical complementary region. It is quite probable that there will be some mismatches between the small RNA and the target, but if they are few they should be tolerated in the hybridization. Anyway, if you find a cleavage site, it may turn up to be worth to switch to a small rna cloning approach.

Thanks a lot. I'll try both ways.
Irina