I will be using different treatments to induce the expression of certain genes in mammalian cell lines (i.e. HEK293, HepG2, and INS-1). I'd like to know how long do the cells need post-treatment to see any changes in their gene expression that can be quantified by Real-Time PCR?
During my RNAi experiments I'd wait for about 48 hrs to see a knockdown effect on the mRNA level, but I'm not sure whether it's the same case for stimulating cells as changes in the expression of certain genes (e.g. genes involved in the cell cycle) take place in a much shorter time.
I think you can use time factor to establish your hypothesis since there are a lot of variations in gene transformation relating to the cell of choice as well as to the treatments involved.
Thus, you may decide to investigate the effect of X compound on the expression level of gene X following Xhrs or Mins of exposure or you may choose to use a repeated sampling approach at different time intervals; X, Y, Z hrs or Mins.
I am not aware of gene changes being reverted to absolute normalcy in short period of time, especially if the compound is still in the media. So there is the possibility of detecting an early gene change even hours later.
I've done some work with a PI3K inhibitor and I see changes by 4 hours. But it depends on your cells and the drug concentration you use. It may be worth doing some experiments with a few time points or drug concentrations to determine what works best for you.
Sulaiman, Without telling us which genes are you interested in and what stimuli you will be using, one can just speculate. The reason I am telling this, and us Yusuf said, different genes are expressed very quickly early time as short as, 15 min for in vitro experiment and 3 hours for in vivo experiment. The best bet is to do a pilot experiment where you choose an early time point; let say 3 hours, and a late time point 24 hours. Then according to the data you obtain you choose the best time that answer the questions or hypothesis. good luck
- Badges
- Science topic