sirna half life - time of effect (May/13/2007 )

hello,


i have gone over many litratures regarding the sirna and am not able to understand something. and would like to ask it here.

if i take a cell line and transfect it with the sirna for a gene, how long will the sirna effect last in the cell. is there any way of prolonging the effect, i have come across the use of virus to infect the cell line with the sirna are these doen because the normal transfection used has a half life time tag.

and is there any difference in the half life time among prokaryotes and eukaryotes.


thekid

Your post title is asking about the life of siRNA, actually you want to know how long RNAi lasts. They are two differnt things.
siRNA is double-stranded, is much stable than single-stranded RNA. When transfected into cells, it exists for a few days.
As to the question how long RNAi (the effect elicited by siRNA) lasts, it lasts only5-7 days. After that RNAi effect will disappear when the trigger siRNA molecules disappear.

To obtain long-lasting RNAi effect, you have to use a shRNA vector which expresses a hairpin RNA. The hairpin RNA will be processed into siRNA by dicer in the cells. You use the vector to establish stable expressing cell.

pcrman is right but the problems with shRNA are that :

1 ) you don't really have any control on the expression of the silencing RNA which mainly depends on your promoter in this case.
shRNA is used to make stable cell lines, so that things tend to be more reproducible once the cell line is selected for its silenced properties,
but siRNA is better suited for a quick and accurate try of silencing.

2 ) shRNA can generate more off-targets than siRNA according to a seminar from a Dharmacon guy.

Apart from that, nobody really knows the lifespan of oligoduplxes used to silence RNA, because it may vary.
Most of time you need to monitor the silencing of your protein to know the effect of your siRNA, but to know the lifespan f your duplex, try RT-PCR at different zimepoints of your silencing 24, 48, 60, 72h seem to be good points, unless your protein is important and its silencing kills.