Exiqon miRNA arrays- any experiences - difficulties in validation (Nov/12/2008 )

We performed a few miRNA arrays using the Exiqon LNA multispecies micro-array chips... I did a total of 6 replicates, so I was hoping to get robust data. Even so, the p-values that we got (we used Genespring to anlayse the scans) were not good.

Now I am using Taq-man assays to try to validate the array results. I have looked at 3 candidates so far, and the array results dont seem to hold up AT ALL!! I am a little flustered!! I am using RNU6B as an endogenous control for the real-time PCR..

Any experiences with either of the two techniques?? Any suggestions would be really helpful!!

Thanks,
Vik.

-Gallus gallus-

I've not used the arrays you mention, but the TaqMan, with Rnu6b, works well for me. I don't know what to suggest, except to talk to their tech support people.

-miRNA man-

QUOTE (Gallus gallus @ Nov 12 2008, 10:38 AM)

I'm using taqman miRNA panel from AB. In my experience using a card gives you a path to follow and validate. usually after a profiling i got around 10 miRNAs which seem to be interesting and they always fall down to 2/3 after validation with single assays. with latest version of miRNA arrays from AB i couldn't even be sure of what was indeed expressed. It could be due to the fact that i used a very low amount of RNA for RT. In my case RNU6B is not a good endogenous control, you should try to validate 1 or more controls for each experiment.
fizban

-Fizban-

Thank you Fizban and miRNA man... .
I wanted to use some other control as well, but the problem is that I am working with chick miRNA's and so only RNU6B has a 100% matching orthologue, atleast as per the BLAST results. So, I am sort of stuck with this control!

But yes, I need to talk to the techinical support from both companies!!
Thanks!

-Gallus gallus-

How is your RNA quality? Make sure not only OD260/OD280, but also OD260/OD230 is good.

I used both, and things work well in my hands. But recently I've got some weird observations with Taqman RT, and it turned out to be the RNA has phenol and/or salt contamination. It makes sense if you consider the reverse transcription primers are in stem-loops..

QUOTE (Gallus gallus @ Nov 18 2008, 12:01 PM)

-yuut-