I have knocked down a gene of interest using siRNA and am using qRT-PCR to confirm reduced expression.
All samples treated with non-specific siRNA have Ct values around 35, but all samples treated with gene-specific siRNA have undetermined Ct values, even after 50 cycles.
I am aware that the gene itself is not highly expressed in this cell line and tried both SYBR green and TaqMan assays. I have also confirmed the knock down of this gene in another cell line with higher expression (Ct around 22). Furthermore, many of the same genes up- and downregulated upon knock down are altered in both cell lines.
It's clear to me that the gene has been successfully knocked down, but I cannot perform any calculations like fold change. Therefore, how do I report these data in a manuscript, for example.
If you want to use these data (even when a Ct of 35 is in my opinion not reliably calculateable / near backgrsound level), you can deterine a Ct of n.d. as 0 in this artificial context, consequently leading to an expression of 0.
On the other hand, I would prefer Western blot or ELISA data to determine inhibitory efficacy of siRNA-mediated down regulation (which represents never ever a knock down). Protein expression data would lead to more reliable results than mRNA expression data (which seems to be rather inadequate following your explanation).
Explanation: In my opinion, siRNAs reduce translation not transcription.
As Andreas said above, a ct value of 35 is negligible expression. I have faced this problem with one gene and I used cq value of 40 (highest cycle) for my KD. Then again if expression is so low, you cannot prove this by WB either. The best way to go is to take the highest cycle number as Cq.
Alternatively, you can still perform an ordinary end-point PCR respectively northern blotting. With an appropriate control that proves the gene expression in control cells without siRNA, you can simply show decreased/absent expression in your knock-down cells (comparable to qualitative western blotting on protein level).
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