I am working with various cell lines and have transfected Luciferease firefly plasmid and Renilla control plasmid for quantification of the signal. However, I am not able to get any reading with the firefly plasmid. I am using 25 ul lysate and 50 ul LAR II reagent. Interestingly, every time I add 50 ul Stop and glow, I get high values for Renilla plasmid. I cant understand if the problem is in the transfection or plasmid expression? I co-transfect ~ 500 ng concentration of both the plasmids together using lipofectamine in cell lines such as MCF7, SHSY5Y. Kindly suggest?
Firstly the renilla plasmid, I assume is the transfection control plasmid and therefore Renilla is continously produced by CMV/TK promoter? This explains why you get a reading for renilla as it is not inducible
The Firefly expression from the plasmid needs to be induced with a stimulus and this depends on the type of inducible promoter controlling expression i.e in our assays this is the transcription factor CREB via cAMP linked to a TK minimal promoter.
You may well get firefly without stimulus but values should be low and perhaps not detectable in your assay. What is you stimulus?
1. Check tfx works, if you get Renilla values above controls then there is plasmid present.
2. Check substrate for Firefly is working or make new stock
3. check your stimulant is working properly
4. Do you have a positive control i.e in our system we use forskolin which stimulates Adenyl cyclase production of cAMP > CREB > Firefly
5. Might be a silly question but do the cells respond to the stimulation i.e contain adequate receptor numbers
6. Are your cells healthy post transfection. Lipofectamine can be toxic?
7. You may need to trouble-shoot the transfection. Usually we transfect in a lot less of the transfection control plasmid ie. 20ng compared to 200 ng of Firefly. All you need from the renilla plasmid is a positive reading so amounts can be a lot lower. There is a possibility that plasmids may not be compatible but without more information this is difficult to confirm
Most likely problem is that you are using too much lysate volume. 25 ul lysate is too much for a (total) 75 ul LARII reaction. Don't use more than 20 ul of lysate for 100 ul of LAR II reaction mix (20 lysate/100 LARII /100SandG)...your ratio is off and once you're past that ratio, values drop precipitously.
Other possible explanations (less likely):
The renilla signal is typically much stronger than the firefly's: your results might reflect low transfection efficiency. Include a positive firefly control (Promega has a bunch of them; pGL3promoter or equivalent has a strong CMV promoter). You should get Mid to high values (10,000 to 100,000 s) for your average lab cell line for 10 ul lysate (10/100).
Are you using their lysis buffer? Makes a huge difference. Home-made recipes probably won't work too well with the firefly's.
Good luck!
The plasmid I am using for firefly luciferase expression is https://www.addgene.org/16442/sequences/ (PG13-Luc). I am not using any stimulus. I dont think any stimulus is required. p53 in the cell will bind to the p52 binding repeats (13 repeats are present in the plasmid) and then luciferase activity can be quantified.
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