Loss of RNAi after changing culture media? - (Jun/28/2007 )

Hello all ,

I know from reading the manuals of various transfection reagents that it is possible to change the culture media on cells eg. +4 hours after transfection with siRNA.

Does this mean that by this time (specific to reagent) all of the siRNA molecules should be within the cells, with none left in the media?

The reason I ask is, to establish knockdown of my gene of interest, I quantified mRNA 48h after transfection, by qRT-PCR. I've observed knockdown to around 20% of untransfected control mRNA level.

In order to test whether my gene of interest is involved in an adaptive response to treatment with a specific chemical, I've transfected with siRNA for 24h, then changed the culture media & added the chemical, incubating for a further 24h.

There doesn't appear to be any diff. between -/+ siRNA. Of course, this may be because the gene is not actually involved in the adaptive response. Also, the 20% of mRNA remaining after siRNA knockdown could be sufficient to enable the adaptive response.

However, I'm curious as to whether changing the culture media 24h after transfection, then treating for a further 24h (ie. 48h total) should give the same RNAi effect as transfecting for 48h without changing the culture media? Could there still be siRNA molecules in the culture media after 24h, that are being removed when I change the media? Is changing the media after 24h the equivalent of transfecting only for 24h?

Apologies if this is a bit of a basic question, I'm relatively new to RNAi. I will probably test the effects of changing the culture media on the RNAi effect, but was wondering what other people's thoughts/experiences were on this subject?

Many thanks in advance for your comments.

-ph0u203d-

Hello,

I think changing medium 4 hrs following transfection is too early and doing that 24 hrs late is fine. We have tracked the fate of labeled siRNA after transfection. in the first 24 hrs, there is still siRNA floating in the medium.

QUOTE

However, I'm curious as to whether changing the culture media 24h after transfection, then treating for a further 24h (ie. 48h total) should give the same RNAi effect as transfecting for 48h without changing the culture media?

I agree. RNAi effect appears usually 12hrs following transfection and remains stable 5-7 days.

Another choice is to first establish stable shRNA transfected cell lines and then do your treatment.

-pcrman-

QUOTE (pcrman @ Jun 28 2007, 06:08 PM)

We have tracked the fate of labeled siRNA after transfection. in the first 24 hrs, there is still siRNA floating in the medium.

Thanks for your reply.

Have you published anything on this? If so, could you provide a reference?

I agree, I think 4h is too early, but I wasn't sure if all of the siRNA molecules should be inside the cells by 24h. You say you've found some still in the media, which is interesting.

Also, if anyone knows of any good papers/reviews on this subject, I'd be grateful if you could point me in their direction.

Thanks again.

-ph0u203d-