workflow - start from microarray - (Feb/07/2009 )

I think many ppl who are new to this field will have similar situation as me

We did microRNA microarray and got some data

But what's next?

I know if you want to prove the relationship between a microRNA and a protein, you can do transfection, then Western, then probably 3'UTR mutation and luciferase assay

But what's the best and most efficient way from microarray data to narrow down one microRNA-protein pair?

Like....what assay should be done to verify the change in microRNA? Northern or realtime? what algorithm to predict target is the best? etc....

Thanks

-jiro_killua-

I did qRT-PCR (TaqMan) to confirm some of the interesting miRNAs on our array and it worked out pretty well and was concordant with the array results. After that, I guess it's up to you and your ideas and what your hypothesis is. My impression is that finding an up/down regulated miRNA and then validating one mRNA target isn't enough to get into a nice journal anymore. I think you need to do some functional studies and see how the miRNA fits into a pathway. But luciferase assay is the way to go to validate targets.

-miRNA man-

jiro_killua on Feb 7 2009, 10:58 PM said:

I agree with miRNA man.
qPCR should be your choice instead of northern, it's much more sensitive and quick.
aside from the "standard" assays such as luciferase and so on you should try to understand or to think what is the real function of your miRNA. having a confirmed target could help, but up to now it is still the hard part of the job!
good luck
Fizban

-Fizban-

You can confirm a target MRE with a Morpholino oligo.

Choi WY, Giraldez AJ, Schier AF. Target Protectors Reveal Dampening and Balancing of Nodal Agonist and Antagonist by miR-430. Science. 2007 Oct 12;318(5848):271-4. Epub 2007 Aug 30.

-Jon Moulton-

Jon Moulton on Feb 10 2009, 07:14 PM said:

not an easy task when your predicted targets are 200/300!

prediction is SO hard!!
fizban

-Fizban-