My protocol is to transfect the fibroblasts for 24hrs and then directly in the next day starvation for further 24hrs and then stimulate with a growth factor. My question is: would it be better to wait 24 hrs after transfection by replacing the transfection medium with serum containing medium and then starve the cells? Is it possible that if I couldn’t obtain 80% knockdown of my protein, the rest of the protein in cell could be further processed?
it's not entirely clear from your question, but I assume you are trying to knock down a target transcript/protein with siRNA? Do you know whether your target protein is efficiently knocked down by 48 hours post-transfection? I would suggest that you first determine the time course of your target knockdown, ideally by immunoblotting or ELISA.
First thanks Andrew for your comment, i tried to knockdown my protein with siRNA for both 24hrs and 48 hrs so I have got approximately 50% knockdown in case of 24hrs and there is no knockdown detected after 48hrs .so i couldn't explain why there is no no knockdown after 48 hrs and what I want to know what exactly happen at the transcriptional level if I knock down a target protein with siRNA for say 24hrs and then let the cells in a rest period and then starve the cells ? and could the long transfection period (48hrs) decrease the efficiency of knockdown as I know that this effect is transient and off target effect could be formed?
Usually 48 hr is not considered long for a knockdown experiment. How are you determining the % knockdown? Have you tried multiple siRNAs? It's possible that your siRNA just does not work very efficiently. Try to optimize the knockdown first before you look at the effect of serum starvation.
I agree with Andrew completely. First determine the the transfection efficiency in time dependent manner. Next as u transfect cells becomes fragile and under stress so u can add serum in transferring media post 5 to 6 h of transfection and incubate for the standardized time for maximum transfection efficiency followed by serum starvayion .
My suggestion is to try all knids of combinations until you get what you expected. Our group once tried a combination of 3 siRNA but saw no change in protein level until we use only one of the 3 siRNA then saw a dramatical knockdown. Why? I have no clue. I also saw rebound of protein level after 48 or 72 hours knockdown, again without any good explanation. I do learn from some article that you do second knockdown but I have not tried it myself.
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