Time course for siRNA inhibition - When is the peak of inhibition after transfection ? (Jun/26/2006 )

Hi, all.
It is about 24 hrs after transfection. I used siRNA against EGFP in a stable 293 cell line with the reporter. Most EGFP signals are gone by now. There are some weak ones left still. How long it normally takes for siRNA to inhibit gene expression to the maximum? Thanks.

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The time is not always proportional to the silencing effect. Some people lyse the cells 24, some 48 and some others 72 hours post transfection. You can check: I think it depends on the siRNA

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The silencing depends on siRNA. For a certain experiment, I have observed maximal silencing after 72 hrs comapared to 48 or 24 hrs. I didnt check silencing after that time point.

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you have to check it yourself, but generally it's between 48 and 96h.

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well, I see no difference at 32 hr post transfection and my luciferase assay indicates a greater than 97% inhibition vs no siRNA group. So I stop here. Thanks all for your suggestions.

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Hi there!

May I ask which sequence you used to target EGFP? Mine doesn't work as well as yours

Thanks, Stardust

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We bought one from Ambion to be used as a control siRNA instead of scrambled ones and if we try to transfect both DNA and siRNA at the same time, it inhibits the transfection (or expression, we don't know) of the cDNAs

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The reporter was pEGFPLuc from Clontech. I ordered 27-mer siRNA from IDT (EGFP-S1 DS Positive Control). This is a Dicer substrate and is ~ 100x more potent than 1st generation 21-mer siRNA, according to published works. The information is in their Dicer-Substrate RNAi Kit section.

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Thanks for the information!

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dnafactory, Just FYI:

I checked co-transfection of this siRNA with pCMVb-gal and found no inhibition on the expression of beta-gal at siRNA dosages that gave ~90% inhibition of its target sequence EGFP.

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