I'm transfecting siRNA using lipofectamine RNAiMAX (at 7.5 uL / well in 6-well plate), into gastric cancer cells (500000 cells/well, in a 6 well plate), and using qRT-PCR assay to test the knockdown efficiency. At the 3rd day, there is no noticeable change in the cell morphology from the negative siRNA compared to the normal cells and significant change in the targeted-siRNA cells (I'm using 25 nM siRNA). But the PCR showed a significant downregulation of targeted gene in both negative siRNA and targeted-siRNA . Has anyone encountered this problem before? Can somebody please explain why this is happening?
Hello Xin Xu,
It can come from many things. And it can also be a problem in your qRT-PCR settings. If you don't use the correct housekeeping gene as reference, you will see a downregulation of your genes, not due to the siRNA.
Which negative control do you use? Do you also have a control of untransfected cells?
I agree with Arnaold. But the "off target effects" might be another reason. Maybe you can try several different NC siRNA sequences. What's more, the cytotoxicty might be another reason. Did you use the lipofectamine as the user guide? Have you observed the cell before the qpcr assay?
May be because of cytotoxic effect of transfection reagent, reduced the incubation time and wash the cells with regular medium,
Gud luck!
Hi
I also agree with Anne above...You need to check with other controls as well as some other genes...down-regulation observed in the control can be due to several reasons (i guess you have also assesses the quality of the RNA preparations too)...It is always advised to use more than one reference genes for such analysis.....and repeat it 2-3 three times
bye
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