Hello everyone,
I am running luciferase assay with pMIR REPORT plasmid to evaluate the function of a miRNA on a specific 3'UTR. I wondering if I could normalize the firefly signal to the number of viable cells counting by Hoechst/PI staining?
Thank you in advance
If you are lysing the cells, then you should estimate the amount of protein by any standard estimation method like bradford. However, if you don't want to lyse the cells then along with your plasmid you should co transfect your cells with another plasmid containing GFP of beta galactosidase gene in a fixed ratio. Then you can normalise the luciferase readings with respective GFP of beta gal readings. This is the standard way to normalise.
I did normalized the data assay with number of cells and I used IncuCyte to count the cells
following on from Abhilash's suggestion, you can co-transfect your cells with a third plasmid expressing Renilla or Gaussia lucifesrase driven by a constitutive promoter- this is a widely used and commercially available plasmid- and use the Dual-Glo luciferase kit (from Promega) which detects both firefly and Renilla signals sequentially within a single well. This will normalise for variations in both cell number and the transfection efficiency across your samples.
Good luck
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