RNAi doesn't work? - (Aug/29/2006 )
I am working gene knockdown by siRNA in human cells. I saw decrease of protein level in immunofluorescence, but I couldn't see decrease in Western blot, and also RT-PCR. Does someone has same problem? I am glad if someone explain this things.
Could u give more details abt the experiment.
How is ur transfection with the siRNA and how long after do u make extracts for western?
R u knocking down some fluorescent protein ? r u using fluorescent labelled siRNA ?
Sorry, i didn't say anything about experiments.
So, detailes of my experiment is follow.
target; transcription factor (protein localize in the nuclear)
siRNA; bought from DHARMACON, we have also siRNA sequence scrambled as negative control.
antibody; homemade (polyclonal), it works IF and WB
Cell line; Hela, HEK293
reagent; Lipofectamine 2000
sampling; whole protein or nuclear extract for WB in 2-4 day after transfection
protein size; about 150kDa
immunofluorescence; 4day after transfection, 1st antibody is homemade, 2nd is anti Rabbit-Alexa488
did u have a transfection control ?
Immunocytochemistry is not quantitative to suggest a knockdown.
If u dont c a difference in WB and by RT-PCR, mayb there is no knockdown or mayb no sufficient knockdown. But one has to consider the half life of the protien as it takes time to c a difference in WB for a protien with a longer half life.
Your observation of decreased amount of target protein by immunofluorescence may be due to general protein synthesis inhibition. WB and RT-PCR are normalized and corrected measurements, thus more realiable. How many siRNA designs have you tested? you may need to test 2-3 different siRNA for the same target to find a potent one.
thank you for many comments.
I had siRNA for GAPDH as positive control, it works in WB.
I also see half life of my protein by cycloheximide treated, the protein was stable, because there were still in the nuclear 1day afeter treated.
I have already tested 5 different sequence siRNA.
I'm preparing stably shRNA expression cell line, and I will test sevral times transfection.