3 replies to this topic

[ucsd]

I using EP get the siRNA into the cells, it works by checking the protein level. However I still could get the mRNA level by QPCR.
the Ct value is simalr to control.
What is wrong with the QPCR or siRNA or my primer?

[cellcounter]

ucsd, on Jul 6 2009, 11:54 PM, said:

I using EP get the siRNA into the cells, it works by checking the protein level. However I still could get the mRNA level by QPCR.
the Ct value is simalr to control.
What is wrong with the QPCR or siRNA or my primer?

I have also heard of this happening to some other people and am curious about it.

Although siRNA may be blocking the protein synthesis, primarily siRNA should degrade the mRNA. The only reasons I can think of your qPCR not working despite mRNA degradation are..

1. You are using wrong housekeeping gene for normalization.
2. You are using primers that bind to an undegraded fragment of mRNA.

These are just thoughts, I have no real knowledge of this. If you don't get any good feedback anywhere, I suggest desiging a qPCR primer pair in another region of mRNA, and/or trying out various housekeeping genes. I would not throw away the cells just yet, since you get knockdown at a protein level.

Let us know if you solve this puzzle.

[miRNA man]

I've not heard of EP, but if I understand your problem correctly, you introduce siRNA into your cells and see a reduction at the protein level, but not the mRNA level. If this is the case, then this sounds very much like a miRNA-like mechanism of translational inhibition, not degradation. Check your siRNA sequence, specifically to make sure nucleotides 9-12 base pair perfectly. If there is a mismatch/bulge then miRNA mechanism is very likely.

Of course, the suggestions put forward be cell counter are also likely/possible.

Did you design the siRNA, or pre-designed by a company? Sometimes they have a qRT-PCR assay specifically for that siRNA.

[Functional Screens]

My suggestion is design a new set of primers for qPCR or qRT-PCR.  These two oligos better probe on two different exons of your target mRNA, then you might be fine.