How to characterize unknown small RNA - (Dec/08/2005 )

Here's the problem: we have a relatively pure mixture of an unknown, short RNA. How do find out what it is? The biggest clue is that the RNA is very probably complementary to a known gene.

I have been thinking about various approaches: RT with unspecific primers, or RT after ligating a known oligo to one or both of it's end. Once converted to DNA many avenues open up - TA clonging, PCR,..

I have also been brooding whether mass spec can be done on short RNA. But I have only seen MS of proteins so far and don't know whether it can be used on nucleic acids. This could work since if I know the nucleic acid composition, I could probably find it through the aforementioned complementarity to the known gene.

However, all these considerations are highly theoratically, that is I don't have lab experience with it. I hope somebody with more experience can advise me as to which avenue to choose.

All the best, j

Hello,

I think the best way of identifying these small RNAs is cloning although I have done it myself. If you want to know if a specific small RNA (miRNA or whatever) is in the pool, you can do Northerns.

You can find miRNA (a type of small RNA) cloning protocols on this page http://www.protocol-online.org/prot/Molecu...croRNA_Cloning/

I haven't done it either, but my first thought while reading your question was to turn it into cDNA and clone it, the same conclusion you came to further on in you post...

It is dependant on your purpose.

If you hope to characterize whole small RNAs, you might clone their cDNA into vector as mentioned by others, sequencing, and character them by blasting with genome/EST sequence. You will find out what they are, such as miRNA, siRNA , other no-coding RNA, even degraded RNA fragments.

If you hope to know whether there are small RNAs(siRNA) come from specific gene, you’d better enrich them by kind of affinity purification (such as using bio-labeled target gene RNA) before clone the small RNA PCR products into TA vector. In such case, you might find small RNAs (sense or antisense) complementary to you target gene.