2 replies to this topic

[FACS_flow]

Hello everybody,

I was wondering if you could also use regular RNA oligos as miRNA mimics instead of buying them "premade".
I am not transfecting these oligos in cells or will study any in vivo/in vitro effects, I will simply
use such oligos for normalization of my qPCR data.
So e.g. I will spike in some c.elegans specific miRNAs for normalizing my human-specific miRNA qPCR.
I just wanna know if someone knows if this works fine as well!
Thanks

edit: I just found this post http://www.protocol-...osts/24008.html
which basically exactly describes my issue. Unfortunately the link provided in this post is dead.

So to update my question: I am wondering if you could use synthetic oligos instead of these synthetic miRNAs (mimics)
for the TaqMan MicroRNA Reverse Transcription Kit provided by AB. I mean miRNA mimics are also only
blunt end, double stranded RNA oligos and that's it, no other fuzz (according to QIAGENs FAQ). And
if you believe the illustration on AB's website about the RT reaction, the stemloop primer is binding to the mature
miRNA, which I will be providing as a synthetic oligo... So what do you think ?

Edited by FACS_flow, 08 July 2009 - 04:52 AM.

[Functional Screens]

Never done this way, but if I am doing it, I will use the mature miRNA sequence (17-24nt single strand RNA oligo) or some irrelevant siRNA (e.g. against luciferase).

[Fizban]

Functional Screens, on Jul 8 2009, 04:51 PM, said:

Never done this way, but if I am doing it, I will use the mature miRNA sequence (17-24nt single strand RNA oligo) or some irrelevant siRNA (e.g. against luciferase).

already done it using mature sequence.
it's ok, it's cheap it works well but BEWARE of contaminations.
i was VERY careful but had to throw away my RT kit anyway.
be very very very careful.
bye
Fizban