I cotransfected HEK293T cells with a plasmid containing 3'UTR of gene of interest and another plasmid containing miRNA sequence. I used equal amount for these two plasmids. After I did luciferase experiment, both firefly and renilla lusiferase activity were reduced in this cotransfected cells. Has anyone had this problem and can it be resulted from the ratio of these plasmids?
In general final results come from Luciferase/Renilla ratio. If you are talking about the following conditions:
A) condition 1
a) 1ug x = 1 ug
b) 1ug y = 1 ug
c) 1 ug x +1 ug y = 2 ug
I assume in this scenario you will see variations in the Luciferase/Renilla RLU
B) condition 2
a) 1ug x + 1ug any similar backbone plasmid= 2 ug
b) 1ug y + 1ug any similar backbone plasmid = 2ug
c) 1ug x+ 1ug y = 2ug
Always keep your Luciferase plasmid same for all conditions, the variation in total plasmids when multiple non-luciferase plasmid are added could be adjusted by additional plasmid with similar backbone
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