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Best time to isolate RNA for qPCR?


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#1 biologg

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Posted 11 November 2009 - 05:43 AM

Hello everybody,

I have the following question, maybe a bit naive: If you add certain substances to a monolayer of cells and you want to check if these substances affect the expression of a gene, when would be the best time after adding the substance to isolate the RNA from the cells for qPCR. Obviously, it is very dependent on the gene, but in your experience with the genes you have investigated - how fast is the gene expression altered by adding e.g. growth factors to the cell culture media and how long is the gene expression kept in upregulated/downregulated state by these molecules? To be more specific, I am interested in investigating the expression of certain genes involved in cell migration and I want to check if their expression is altered by adding different growth factors to the cells. I will obviously have to isolate RNA at different time points. But I was just thinking - whether it would be wise to start the RNA isolations from 1-2h or from much later timepoint - let's say 6-12h after adding the growth factors? What would you do?

#2 Admiral_Stuckov

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Posted 13 November 2009 - 06:34 PM

I am dealing with vitamin D-induced anti-proliferative effects and usually I see the most significant gene induction in the range of 8-24h depending on the gene. I think you could use a control for your gene of interest to see when it is expressed, but a good reference point would be 16h (at least for my type of experiments). Cheers




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