Why most researcher run the viability assay after 24 hr of treatment not 48 hr or 72 hr.? To be specific I am talking about treatment of human cell lines with liposomes carrying siRNA.
Most of the research papers demonstrated the cytotoxic effect on human cell lines after 24 hrs of treatment with liposomes
Most of the cells divide every 24 h, and thus incubation for 72 hours will result in cell proliferation by 3 times. Given that cells were transfected with siRNA only once, after 72 hours of incubation the siRNA will be diluted as a result of cell division. Therefore, percentage of cell viability is expected to be increased (cell toxicity will be reduced) due to siRNA/liposomes dilution caused by cell division and this explanation is consistent with previously reported paper by Bartlett DW et al.
Bartlett DW, Davis ME. Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging. Nucleic Acids Res. 2006 Jan 12;34(1):322-33.
https://www-ncbi-nlm-nih-gov.uml.idm.oclc.org/pubmed/?term=Insights+into+the+kinetics+of+siRNA-mediated+gene+silencing+from+live-cell+and+live-animal+bioluminescent+imaging.
The answer to your Original question is in the paper itself- see Fig 2. The maximum silencing (least signal intensity for luciferase) is around 72 h.
What is not told in the paper is the half life of proteins already synthesized. While silencing with siRNA will be diluted due to cell division- the silencing is usually sufficient to keep the concentration of proteins to a minimum even at day 3.
For practical purposes, if you want to look at the concentration of mRNA then your methodology should be different as opposed to when youre looking for a read out that is dependent on the concentration of the protein. This would be best achieved when the protein concentrations are the lowest.
One way for you to find out the kinetics of YOUR fav protein (YFP) is to silence cells with siRNA (but make sure you sue controls to calculate transfection efficiency) and to extract proteins at regular intervals and run a western to determine the concentration of the protein and check what time point corresponds to the lowest protein concentration. Then perform viability assays at that time. That would be the MOST representative method of figuring out the effects of viability wrt to (YFP).
Hope this helps; if you need to chat- come by you're right next door dude.
:)
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running