2 replies to this topic

[Bomber]

Hi all!

I hope somebody can give me some input here -
I am using two different siRNAs (even from different companies) trying to knowk-down my gene of interest, for which
I don't have any convincing antibody around.

Transfecting these two siRNAs into the cells results in a phenotype which I can observe with both siRNAs (though to a different degree). The phenotype is also present when unsing 5nM siRNA (tested for one of the two siRNAs - which I think is pretty low).
However, I repeatedly failed to demonstrate a knock-down of my gene via RT-PCR, which is weird and frustrating :-/.
So now I am thinking about the following -
everything I see is just bullshit and I should stop the project....though I don't really like this idea
or I try yet another siRNA - this time I was thinking about smartpool from Dharmacon as a last resort.
Is it possible that mRNAs supposed to be cleaved by siRNAs are maintained in the cell and therefore I don't see any efficiant knock-down?

Any input really appreciated here!
Thanks a lot in advance!
best regards,
bomber

[kylvalda]

Hi bomber,

just two quick questions:

1)you do not see the phenotype with the non-silencing control?
I assume you are incubating with some sort of serum-free medium which already has an effect on the cells
and do you have a positive control siRNA like GAPDH or Cyclophilin B?

2)how do you do the qPCR?
you are sure that everything is going fine there:
primer specificity if using SybrGreen is okay?
do you do an absolute quantification with standard dilution series or just ddCts?

kylvalda

[Bomber]

hi!

thanks for your message - nope, the phenotype is not there with the non-silencing siRNAs; also here I use two different ones (according to company)

I don't use a positive control but I have knocked other mRNAs in the same cells usig the same transfection procedure; also I know transfection efficiancy is pretty good (using an labeled siRNA control)

I am using ddCTs values for quantification; primer design was not so simple but looks ok now - but I am really wondering if I should design the primers in such a way that they would amplify the part where my mRNA is cleaved - I am really wondering if cleaved RNAs may reside within the cell and therefore give me no knock-down on RNA level.
Have u ever heard about this?

Best regards!