siRNA concentration in divinding-growing cells - (Mar/22/2009 )
Hi all,
I would have a question regarding siRNA transfection efficiency. We use Lipofectamin 2000 (Invitrogen) for siRNA transfection. There is written in the working protocol that cells should be 30-50% confluent at the day of transfection (to avoid overgrowing cells in several days after transfection - cells can grow 24-96h after transfection). We think that efficiency of transfection is almost 90%, i.e. 90% of cells are transfected.
My question is:
What is happening with siRNA in the cells when these cells are dividing (growing)? For example, if one cell divides in two cells, is any siRNA in the “second-new” cell - does concentration of siRNA decrease about 50% because one cell dives in two cells or siRNA stays just in one of the cell and the “new” cell does not contain any siRNA?
Or in other words. If in the day of transfection, cells are 50% confluent and 90% from these cells are transfected, and after 48hours cells will be 100% confluent there is probably strong decrease of siRNA concentration in cells and siRNA effect is also decreased, is it right?.
Thanks for advices.
Vic
From Tom Tuschl (http://www.rockefeller.edu/labheads/tuschl/sirna.html):
Depending on the abundance and the life time (or turnover) of the targeted protein, a knock-down phenotype may become apparent after 1 to 3 days, or even later. In cases where no phenotype is observed, depletion of the protein may be observed by immunofluorescence or Western blotting. If the protein is still abundant after 3 days, cells need to be split and transferred to a fresh 24-well plate for re-transfection. In the case of the lamin A/C knock-down (lamin A/C is not essential), we monitored silencing after 44 hours, but the knock-down of lamin A/C persisted for more than 105 hours (6 generation times), and the protein levels returned to normal levels only after 170 hours incubation (10 generation times). This indicates that replication of siRNA duplexes may not occur in mammalian cells. It also appears that silencing does not spread to neighboring, non-transfected cells.
I think that there must be a huge excess of siRNA in freshly transfected cells, which gradually diminishes as the cells proliferate. Just my assumption though :-)
miRNA man on Mar 24 2009, 12:38 AM said:
Depending on the abundance and the life time (or turnover) of the targeted protein, a knock-down phenotype may become apparent after 1 to 3 days, or even later. In cases where no phenotype is observed, depletion of the protein may be observed by immunofluorescence or Western blotting. If the protein is still abundant after 3 days, cells need to be split and transferred to a fresh 24-well plate for re-transfection. In the case of the lamin A/C knock-down (lamin A/C is not essential), we monitored silencing after 44 hours, but the knock-down of lamin A/C persisted for more than 105 hours (6 generation times), and the protein levels returned to normal levels only after 170 hours incubation (10 generation times). This indicates that replication of siRNA duplexes may not occur in mammalian cells. It also appears that silencing does not spread to neighboring, non-transfected cells.
I think that there must be a huge excess of siRNA in freshly transfected cells, which gradually diminishes as the cells proliferate. Just my assumption though :-)
miRNA provided us very useful information of the stability of transfected siRNA. siRNA and plasmid could not equally seperated into two daughter cells, maybe by chance, since they were not intergrated in to the genome. More important, they would be degraded as time goes by. But the time is enough for your functional experiment, otherwise siRNA had been gived up. Just my assumption also. May it help you and good luck.