pre-miRNA existence - How to confirm a pre-miRNA exists? (Jan/26/2010 )

I got one new mature miRNA by cloning and sequencing. Now I want to make sure the pre-miRNA exists, but I don't know how to do that. Does anybody have suggestions? Thanks a lot.

j123 on Jan 27 2010, 12:31 AM said:

You have 2 ways:
1) you can use in situ hybridization with probes specific for both pre and pri miRNA. LNA probes probably better. i saw some very interesting results in cambridge 2 years ago, you could have surprising results, keep us up to date!

2) qRT-PCR. This is a littlebit tricky. Precursors have hairpin structures so they are not good candidates for primer design. u have to cope with it. i managed to design working primers for my miRNA of interest, it took some work and stratagene Herculase to work with complex regions, but its feasible.
if i remember well it's possible that Qiagen sells PRE miRNA ready primer sets but u have to check.

probably others have better ideas but that's all i can think now.
i'd not try northern blot, it could be a waste of time if your miRNA is not strongly expressed but this is a personal opinion.
good luck
Fiz

Thanks a lot for your helpful suggestions. Actually, I tried northern blot, but it didn't work for me. For the qRT-PCR, because my miRNA is a new one, I am wondering if there is custom service for Taqman probes in Applied Biosystem. I called them, but I haven't got any response yet.

Fizban on Jan 27 2010, 04:22 AM said:

j123 on Jan 27 2010, 04:12 PM said:

Here in italy custom probes design and validation is available. you have just to wait for validation of your probes but shouldn't take long.
If it works in italy....it works everywhere!
sad but almost always true
bye Fiz

Hi, Fizban, could you please send me the link? Thank you very much for the helpful discussion.

Fizban on Jan 27 2010, 11:47 AM said:

news from ABI: they have PRI-miRNA probes!
https://products.appliedbiosystems.com/ab/e...mp;catID=607040

Actually there's no link for mirna custom assays on the website, just contact your local ABI representative and ask. they have stock of inventoried assays and made-to-order assays. you give them the sequence and they'll give you a VALIDSTED mirna assay. that's how it worked for us with probes made for zebrafish.

hope it helps
Fiz

Yes, they can make customerized Taqman probe for you. It costs about $230, and a week's wait. Use their website for ordering.

j123 on Jan 27 2010, 09:12 AM said:

Thanks a lot for the information. I really appreciate it.

I check AB website, there are two kinds of probes :
* TaqMan® MGB Probes
* TaqMan® TAMRA™ Probes

Could you please tell me which one I should order? Thanks again for your kind help.

cathywill on Jan 28 2010, 02:36 PM said:

I ordered the MGB probe with FAM Dye, since it binds to the loop part of the pre-miRNA. It is a hydrolysis probe which will give out signal at the elongation stage of the PCR.
Of course you will need sense and antisense primers binding to the 5' and 3' end of the pre-miRNA sequence, respectively. Check the guideline for primer design. They have relative strict rules for the sequence and Tm for both primers and probe.
Not familiar with TAMRA probe.

j123 on Jan 28 2010, 02:59 PM said: