microRNA Cloning - Advice...anyone...please! (Nov/23/2005 )

Hey Guys,

How are you all?

We are looking to isolate and clone miRNAs from the differentiated primary cells we are interested in with the very slim hope of isolating a cell type specific miRNA. The problem...I have absolutely no experience in this and was hoping to get a little advice?

I have obtained a protocol from the Bartel Lab which is thankfully very detailed but there does seem to be an awful lot of radioactivity involved! I have also looked in to the seemingly quicker Ambion protocol (TechNotes 12(3)). To me it appears that the Ambion protocol os more or less based on the Bartel protocol with the variation of pushing their own products for steps 1 & 2, after which they get quite vague. I am assuming the subsequent steps are bascially the Bartel protocol and therefore requires radioactivity but I am not sure. Has anybody done any miRNA cloning using either method? Is there anyway I can perform this without using radioactivity (as you can probably tell I REALLY don't like using radioactivity..what a wimp!)?

Anyway, sorry for waffling and probably coming across as a very stupid young lady indeed!

Have a good day/evening wherever you may be.

Thanks in advance for any help.

Holly

We used 10bp DNA ladder load in other lane, then cut gel of RNA lane corresponded to 15-35bp range of DNA maker…Although the move speed of DNA maker might differently from RNA, and the cutting size of RNA might not precisely as Bartel protocal, but it work.
good luck
Rui

Hi,

you can purify similar to how Rui describes, but instead of referring to a 10bp DNA marker, you can cut out a band corresponding to the lower two thirds of the gel between the bromphenol blue and xylencyanol dyes. This corresponds to RNA below 60-40 nt in length.
You should use a small high percentage denaturing gel. In order to keep RNA-loss low, try to work with small gel slices (e.g. do not allow nucleic acid seperation to be too large = short gel run).
To check whether you cut out the right band you can use a marker in a seperate lane (10 bp ladder) and stain the gel with ethidiumbromid after cutting.

Hope this helps,

Vera

Hi

Following paper describe another method for cloning microRNAs, which based on polyadenylated RNA techniques. It seem pretty easy, disadvantage: need to figure out 3’ end if cloned microRNA end with A residue.

Identification of human fetal liver miRNAs by a novel method.
FEBS Lett. 2005 Jul 4;579(17):3849-54.

hope it help you

Rui

Hi, there;

I have tried to enhance mciroRNA using kit from Ambion.com and then cut the bands of microRNA around 17-26bp.It works. But I have also tried just to run the total RNA on 15 PAGE gel and then cut the simialr size bands. It still works. The key point is that you should use a 10bp marker to decide the size you should to cut. I tried both EB and Gel-star to stain my gel. They work! But Gel-Star gave me much better stain than EB. The best thing is that you can handle the staining on your bench if you use Gel-Star.

The bad thing is that you can not see the RNA ranges around 17 to 26bp easily. But you can just can the bands and go to eluting step to get you microRNA out.

Good luck!

David

Hi, I used the protocol
Identification of human fetal liver miRNAs by a novel method.
FEBS Lett. 2005 Jul 4;579(17):3849-54.

It works very well for me, I sequenced about 400 clones, and a half of them are miRNAs, including 4 new one, it is relatively easy. But one clone contains just one miRNA sequence, and need to rule out the possibility that miRNA end with A, so you need to check back the precusor sequence. By strictly picking up 109bp inserts (bacteria PCR, run 2% gel), you could have more chances to sequence miRNA. another thing I found that, the RT primer with 1 random nt at 3' end is better than 2 nt. Good luck.