Looks like unequal transfection. Calculate the total quantity of DNA that is being transfected into each group and keep that equal. If required use some 'dummy' DNA/irrelevant plasmid that cannot be transcribed to equalize the transfection quantity. Use similar amount of transfection reagent also between all the groups

What is the value of Renilla in your positive and negative controls? Is it possible that you are getting inhibition in your GG samples that is lowering your IC (Renilla) levels?

Dear Kimberly,

The value of renilla in negative control is 1300 and in postive control is 2400.

Maybe also the way to prepare your transfection mix. Do you make a total mix containing the renilla plasmid. Then you distribute in individual tubes where you finally add your specific luciferase vector?

Renilla is 60% GC so is fairly GC rich. Perhaps there is preferential binding for your GG test sequence and thus the Renilla is unable to be amplified as much as it is when paired with your AA sequence. Have you tried increasing dNTPs?

Dear Kimberly,

I partly got your point. What does increasing dNTPs means. Does it mean to use dNTPs in the lipofectamine-DNA mixture?

Hi Sailesh

I t is quite common to see some discrepancies  in transfection controls but 20 X is quite a bit...some of the previous points are important (co mixing, equal amount and DNA quality et.c..)

What do your GG and AA luciferase numbers look like? Are we talking millions or thousands? Are they fairly similar?  I can think of possible squelching effects by your GG construct on your renilla construct (e.g. titrating away transcription machinery  components away from your renilla construct). Good luck!