Transfection of HeLa cell - any advice? please help! (Feb/24/2005 )
Hi there, please can someone help!
I am trying to do transient co-transfections using HeLa cells and FuGENE 6 reagent. My luciferase reporter construct (in pGL3-basic) is co-transfected with the firefly control reporter driven by sv40. I do these is 96-well plates.
The problem is that I am not really getting any consistent readings even when i do exaclty the same protocol. I have checked DNA purity, for cell infection and all seems fine. I get very dismal numbers for the firefly readings and ok figures for the control renilla. Does anyone do 96-well transfections using HeLa cells that could help? how much DNA should i use/well? Currently i transfect 100ng DNA/well(+12.5ng control vector).
Any advice much appreciated 
hi
you can see the discussion at
http://www.protocol-online.org/forums/inde...?showtopic=5400
Fred
Thank you!
If I understand correctly the fact that you are getting a signal from the SV40 driven renilla plasmid and not your pGL3 construct (which I assume contains a promoter that you are studying) means that the transfection is working. The fact that the siganl is poor for pGL3 could mean that the promoter under investigation has weak ativity in your cell line. Have you tried increasing the amount of plasmid?
Thank you for the suggestion-
I will try increasing the amount of plasmid DNA and will hopefully see an improvement.
I think what worried me the most was the fact that occasionally, in some of the duplicate wells, i get a good signal for pGL3; whilst in other (identically treated) wells I get very low signals.
Do you think the confuency of the cells the day you passage and seed them have a factor in terms of their overall uptake of the plasmid? Currently i grow them up about 80% confluency and then passage and seed them in 96 wells the day before transfection.
either way, I guess its a case of trial and error.
I have tried upping the amount of plasmid by two fold; still hasn't helped.
Is it ALL to do with the exact number of cells in each well? I incubate for 48hrs before reading plates.
hi
maybe the ratio fugene 6 / dna is wrong? is it a good agent for hela cells?
i have take a look at the lipofectamine protocol. And it seems that for a 96well plate you should use 0.05 to 0.1 µg of DNA
i explain:fo 24 well plate they recommend 0.2 to 0.4 ng
so 96well plate : surface is 4fold less
so if we adjust DNA quantity it is 0.05 to 0.1µg
hence i suppose thez quantity of dna is correct...
regarding your seeding protocol, i think it's ok
Hello
I have performed transient transfection with HeLa during my thesis 
. It was with Superfect reagent :
"http://www1.qiagen.com/literature/handbooks/PDF/Transfection/TF_SuperFect/1023348_HB_SF_1202.pdf"
It was also in 12 wells never more wells. The quantity of DNA was equivalent of 1 ug and the ratio of DNA/superfect 1/2 (however 1/3 is also good, in the handbook, it is advice to use 0,5 ug of DNA. )
The medium of transfection was DHG WITHOUT serum (it drastically reduces efficience)
After transfection, the cell rested during 20 hours in DHG with 10 % serum AB. I suppose that you stimulate the cells too, the better condition found (I can't use "past " in english sorry fot the mistakes) was 28h in DHG+BSA+antibiotique.
For the firefly, if your RLU are too low, diminish the concentration of renilla. for example 1/50 of renilla and 49/50 with firefly.
bybye 
HiYa Shojjahd,
A couple of things ...
a) FuGene works beautifully in HeLa but you need to determine the amount to use for your transfection. For a 96-well plate, typically 1.5 µl per well of fugene is all you need.
the confluency of your cells is important. Keep them ~70% confluent when transfecting, especially if you incubate the cells for 48h post-transfection.
c) Do you prepare a master mix (Supermix) of DNA + Fugene in serum-free media before adding to cells?? This is crucial to keep some consistency.
d) And you are using 100 ng total DNA ... this is a lot for a 96-well plate. The suggested amount is 30-60 ng total, and usually the main reporter gene is 50% while the Renilla luciferase is at 10% of the total. You can used salmon/herring sperm DNA to standardise the amount of total DNA.
e) How fresh is your luciferase substrate??? If using the Promega DLR assay, do not freeze thaw more than three times.
Hope this helps.
Hi there
You seem to be getting great advice, but I would mention one additional thing for you to consider. I was routinely using the dual luciferase assay for my PhD work and found that the renilla control construct is able to 'squelch' the activity readout of the firefly luciferase. You may want to try decreasing your amount of co-transfected control vector. In our assays we were using as little as 10ng per 100cm dish of a TK promoter-driven renilla. I expect that you may see a subsequent increase in activity from your pGL3-based vector.
Hope this helps, and good luck.
A couple of things ...
a) FuGene works beautifully in HeLa but you need to determine the amount to use for your transfection. For a 96-well plate, typically 1.5 µl per well of fugene is all you need.
the confluency of your cells is important. Keep them ~70% confluent when transfecting, especially if you incubate the cells for 48h post-transfection.c) Do you prepare a master mix (Supermix) of DNA + Fugene in serum-free media before adding to cells?? This is crucial to keep some consistency.
d) And you are using 100 ng total DNA ... this is a lot for a 96-well plate. The suggested amount is 30-60 ng total, and usually the main reporter gene is 50% while the Renilla luciferase is at 10% of the total. You can used salmon/herring sperm DNA to standardise the amount of total DNA.
e) How fresh is your luciferase substrate??? If using the Promega DLR assay, do not freeze thaw more than three times.
Hope this helps.