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Determining the amount of plasmid transfected


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#1 elray152

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Posted 05 April 2013 - 07:56 PM

Hello, I would like to perform a dual luciferase assay for miRNA binding. First, I would transfect my cells with the two luciferase plasmids, afterwards I would transfect cells containing luciferase plasmids with my miRNA expression plasmid. The question is how do I quantify the amount of plasmid in the cells? The miRNA plasmid has a GFP gene and is under the control of a H1 promotor. Would it be better to try this with a lentiviral vector or perhaps try establishing a stable cell line? Thank you!

#2 bob1

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Posted 05 April 2013 - 11:43 PM

There is no way that I know of that will tell you the amount of plasmid in a single cell from a population other than isolating that cell and doing something like qPCR. However, this would preclude you also doing a luciferase assay on the same cell.

For a cell population you can measure the amount of plasmid by qPCR or by fluorescence signal, so long as you have some standards. Establishing a cell line is problematic,as unless you have a means of doing a single insert, it doesn't solve the problem you currently face. Also, the effect of the miRNA or gene of interest may affect how the cells bind to a surface and/or grow.




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