I do realtime PCR for a target protein after doing siRNA transfection. However, every time when I run real time PCR, the mRNA level of target protein is higher than in the control siRNA. In the fist time, I thought I made mistake of labeling the samples but second time, I have the same data.
Do you know why I have a very weird result? Is it possible the primer is the reason for this weird result?
Thank you!
I do electroporation with NIH3T3 cell lines. I dont use stable cell life. I over-expressed the gene conjugated with GFP and co-transfect with siRNA, I observed a depeletion of my target protein. It means that the siRNA works well.
Today, I tested with other gene and observe a strong signal for the control gene. It means the primers has problem and I need to design another primers for this experiment. The primer is designed by a a software online. However, I still dont understand why the control siRNA showed lower expression. Even though the primer has problem, it supposes to have equal expression at least.
Anyways, for a primer that can detect other gene could not conclude anything at all.
Thank you Kamil!
I bought siRNA form comercial company, Santa cruz.
I looked at the cell migration and morphology of cell but I did not observe any difference in cell migration and morphology between control siRNA and target siRNA.
I could not use western blot because I could not observe the endogenous of target protein so I have to use RT-PCR. Northern blot is very good way to test mRNA level but I could not do in my lab.
My lab, we have lipofectamin 3000 and RNAiMAx. I might try one of them to do siRNA experiment.
Thanks Andrei!
Hi,
Did you check for the hybrydation curves after the dehybridation step at the end of your PCR? And which range of Ct do you get?
As other people already said, is difficult to give a certain explanation because we don't know the whole details of your experiment, I was wondering if you included a not-transfected control. It may be possible your scrambled siRNA is affecting other regulatory elements in the signal pathway in which your gene of interest is involved, promoting the opposite effect that you expected. Although less likely, but the commercial scrambled controls may have off-target effects depending on the cellular context.
First of all I do not understand your scrambled control. The overall meaning of a scrambled siRNA control sequence is that it has NO target gene at ALL! The scrambled siRNA I use has only 31 partial (
Thanks Volker very much for very detail instruction. I used GAPDH or Actin as normalize gene. I will learn more REST2009.
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