PCR for checking transfection - (Jan/19/2007 )
I've read that some people use just PCR to assess that the transfection efficiency of a stable cell line hasn't decreased with the time (if you get the same intensity, you assume that the transfection is still equally effective).
I wonder if it is possible to PCR with primers specific for your plasmid without extracting RNA from your trasnfected cells. To say, you get a batch of cells, centrifuge them, eliminate the cuture medium, heat to 100C for 10min and then do it a normal PCR. The presence of your plasmid should be enough to be detected doing this simple method.
I know that other methods like immunoassay should be used to assess expression but for a start I think this is a good approach.
Anyone has done this? Any suggestions? Is there any problem in my thoughts?
Any idea or suggestion, pleaseeeeeeeeee?
DNA PCR will tell you that the plasmid is still there (or at least parts of it), but it doesn't tell you wether your gene is still expressed.
I haven't performed your procedure, but it is comparable to colony PCR procedures for checking bacterial transformants, so it should be OK if your PCR conditions are optimal.
But if you are culturing cells with selective pressure, the plasmid should be there anyway, regardless of expression of your gene of interest, so I don't know if your check would be usefull?
Maybe others know more about this?
Why don't you try westernblot from cells you wish to verify expression. It might be a better way to check for expression over period of time.
Thanks for the replies. What vairus says it is exactly what I want to do. I know that this doesn't mean that my gene of interest is expressed but it gives me an idea that the plasmid is present and the resistance to the antibiotic is not due to something else like "evolution".
I agree that Western Blot is probably the most common and easy way to assess that my protein is express. In my lab, western blot isnt one of the everyday techniques, so I was looking for an alternative method (less reliable, I reckon) However, in my case I haven't been able to find comercial antibodies for one of the proteins I am interested in. I should look more carefully.
More reliable would be to isolate RNA and see if your gene of interest is still expressed on RNA level. Still not the same as blotting, but more reliable than checking its presence by regular PCR.
Thanks various. I take note of that and probably sooner or later I will have to do that way, RT-PCR.