Hi all,
I am working on dual luciferase assays using Dual-Glo kit (Promega). My experimental vector is pMIR-report (with my inserts cloned into the MCS) and control vector is pRL-TK. The ratio of my experimental:control is 5:1 and I transfect a total of 13.2ug of DNA (11ug experimental + 2.2ug of renilla; cotransfection), using Polyplus Jetprime reagent. My readings are in triplicate. I am seeing huge variability in my renilla luciferase values, example: 8440, 105387, 329664. Firefly luciferase values are fairly uniform, example: 13267000, 12537200, 11955000. I am confused as to what is causing this huge variation in renilla values? Is it my experimental:control ratio? I have to do a rapamycin treatment in the 96-well plates and for this I have to replace the media in the wells with a low-serum media. Am I losing any cells during the aspiration (although I am very careful and trying not to)? Help me!
Dear Shruthi,
may I ask some questions before thinking of an answer? How often have you performed the experiment so far? Which cells do you use and how do you count and seed them? Do you vortex your vectors extensively? Why do you use such a huge amount of vector? Readings are already pretty high...
Regards
Boris
I agree with Zhang, that is a high amount of vector DNA. However, if you look at the data Shruthi provided, she is still within reading range.
B
Boris Schmitz , i have done this experiment definitely more than ten times. I use HEK293 cells, i count them using a hemocytometer. i seed them into 96-well plates, by mixing them well in a reservoir for cell seeding. i dont vortex my vectors, but pipette them up and down (mix them by pipetting). so, i plate cells into 10cm dishes (~2million cells/plate) and transfect them. After letting the cells recover overnight, I replate these transfected cells into 96-well plates. After letting them attach and recover overnight, I replace the media with serum-free DMEM and I do my cell treatments and dual luciferase assays (using promega dual glo kit) within the 96-well plates. Earlier, i worked with 10:1 co-transfection ratios of experimental vector: control vector, in the same cell line. However, the renilla readings for my reporter genes, according to my mentor, were not that significantly above the background non-transfected cells. (Promega tech support, however, say that they're well above the background). Hence, I started using more amount of renilla vector to get a reading well above the background.
Hi Shruti,
concerning the vectors - you should definitely vortex them! And I mean vigorously for at least 1 minute or even longer! If you use a nanophotometer to check the concentration you will see significant precipitation after a short time and you will have a random concentration of vector DNA if you don’t. I would recommend to seed the cells directly into the 96-well format (why the 10cm dish step?) and transfect them. However, for optimization, you might want to start with the 24-well format and then scale down. Also, if it is not absolutely necessary, don’t starve your cells! Or if necessary only go down to 0.5% FCS. I would start over and try different combinations of your firefly/ renilla vectors. In the 24-well format, 1 µg should be enough! So try 1:1, 1:0.5, etc. AND you might want to check your vectors. So try separate transfection to see if your preps work. Maybe prepare new preps (only use endotoxin-free maxis and never mini preps and start from the original stock) and maybe digest and/or sequence your vectors. Use a control vector (pGL3_ctrl or else) to check you transfection efficiency. And always do what you mentor told you ;)
Boris
Boris Schmitz Thank you for the troubleshooting options! The reason that I am transfecting the cells in 10cm dish and then replating them into a 96-well plate, to avoid a washing step to remove the transfection reagent from the 96wells (after the incubation period), to prevent cells from getting washed away. But, is it necessary to wash the wells with PBS to remove the transfection reagent (after the incubation time)? My next step after transfection and replating is drug treatment and detecting the reporter activity using Dual-Glo luciferase assay reagents.
Hi,
you do not need to wash with PBS! Just remove the medium with transfection reagent carefully and then add new medium. If you really are loosing cells (which I doubt) you might want to try iron supplemented FCS for the HEKs. However, I would transfect in the target plate (no additional step), remove medium after 4h+ (transfection should be completed after 4h) and add fresh medium carefully (maybe already prepare your drugs in the fresh medium). If everything else is fine ( efficiency , vectors etc.) it should work.
Boris
Boris Schmitz Thank you for clarifying! I'll try to get rid of the additional step. I'll let you know how it goes :) My firefly luciferase vector is pMIR-report (invitrogen) and the control vector is pRL-TK (promega). I've optimised (or so I think) 5:1 ratio of firefly:renilla vectors. However, promega recommends NMT 10:1 cotransfection. I am trying to scale it down to 10:1 today and transfecting 9ug : 0.9ug of my firefly : renilla vectors. I hope this provides good signal (not too saturating or not too low). Fingers crossed!
Boris Schmitz, Hi Boris, Thanks for checking on me! Unfortunately, I had to pause the Dual luciferase assays for a while. Hopefully, someone else will take over the assays in the future.
- Badges
- Science topic