Knockdown detected by western but not RT-PCR - (Feb/07/2007 )
Hi,
I've been doing some siRNA experiments in mouse cells in culture and I can detect knockdown of my gene by western but when doing RT-PCR my transcript levels seem the same as controls. My RT-PCR is semi-quantitative (i.e. number of cycles used in PCR is in the linear part).
Has anyone else come across this or does anyone have any possible explanations?
Thanks
-bagpuss677-
One possibility is that your siRNA may not exactly match the target sequence and this can lead to inhibition of translation.
-mateo-
QUOTE (bagpuss677 @ Feb 7 2007, 10:17 AM)
Hi,
I've been doing some siRNA experiments in mouse cells in culture and I can detect knockdown of my gene by western but when doing RT-PCR my transcript levels seem the same as controls. My RT-PCR is semi-quantitative (i.e. number of cycles used in PCR is in the linear part).
Has anyone else come across this or does anyone have any possible explanations?
Thanks
I've been doing some siRNA experiments in mouse cells in culture and I can detect knockdown of my gene by western but when doing RT-PCR my transcript levels seem the same as controls. My RT-PCR is semi-quantitative (i.e. number of cycles used in PCR is in the linear part).
Has anyone else come across this or does anyone have any possible explanations?
Thanks
With the western, one cannot be really quantitative. Manytimes the control and sample ones could look different, but infact they migh not be different. How much different was the control and treated samples by western.
But if you saw an all or none effect, then its more acceptable.
-scolix-
Maybe your siRNA duplex is not good for loading into the RISC (thermo stability consideration), and the RISC select the other strand as the functional one. And luckly, that one has a sim-miRNA effect in the 3'UTR you selected...
-MicroRex-