Increment of mRNA level after siRNA

Jose de las Heras via methods%40net.bio.net (by josenet from tiscali.co.uk)
Thu Jan 31 15:22:31 EST 2008


"Kamalian, Laleh" <L.Kamalian from liverpool.ac.uk> wrote in message 
news:mailman.435.1201797462.2451.methods from net.bio.net...

Hi
Thanks to people who helped me last time.  This time its not a desperate 
question.  Only to ask your opinions.  I have successfully used a plasmid 
vector to subside the mRNA level of the gene I'm working on.  I used 4 
different oligonucleotides to insert into the plasmid and used them to 
target my gene.  Although most of the plasmids have successfully decreased 
the mRNA level (different levels of course), one of the clones has actually 
increased the level of the gene's mRNA.  I can't explain it.  Some of my 
colleagues suggest the compensatory mechanisms of the cell.  I think its 
simply not being knocked down or maybe an error in real time PCR? I had seen 
this before working on another cell line.  What do you think?
LK



I think that you you probably need to pay attention to your controls. You 
start saying that the mRNA levels go up after transfection... but then you 
say that you think maybe it's just not being knocked down or an error on the 
Q-PCR.

If I'm just after knocking down a gene, I try 4 oligos, and 3 work... I am 
very happy, ignore the 4th oligo and proceed with whatever I had in mind 
using the other three ;-)

However, if you think that the fourth one is actually increasing the 
expression, depending on what you're studying, it may actually be 
interesting. So I'd ask myself: am I interested enough to pursue this? If 
yes, I'd want to repeat it. If the effect is real, it'll happen again. If 
not...
But if you're having doubts whether it goes up, or it's just not being 
knocked down, it suggests that teh change is very small. Is that true? A 
small change may still be significant, but you have to look at how variable 
your data are... is the increase within the normal range of variation in 
your experiments, or clearly above that? You say you can't rule out a PCR 
error either...

I guess you need to repeat that experiment anyway, to confirm 
reproducibility... for both the bad oligo, and the good ones.

Jose
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