I am transfecting these genes in MCF7 cells and try to evaluate the killing efficiency of the gene of interest?
Thank you Arjan for your reply, yes the vector is empty, we are using lipofectamine from invitrogen for transfection and the efficiency is 70%-80 % most of the times,
Maybe a few naive questions:
Do your genes of interest actually do kill the cells?
Did you include a positive control that is known to kill cells upon transfection?
Technical points regarding the cell cultivation and the details of the MTT assay:
Do you cultivate the cells long enough to see differences (if there are any?)
How were the optical densities at the read out? Ideal ODs are at 0.1 for blanks and 0.2 to 1.0 for actual measurements; above and below results are not reliable in a optical UV-Vis spectroscopy reader - in my humble opinion.
@Alexander: Thank you. Yes we suspect that they kill cells, Other studies shows that .. moreover we are using p53 as positive control. We are using MCF7 cells and after transfection cultivation time is 48-72 hrs.. Here I am attaching a data list of them .. please let me know if you have any other suggestions..
Hello Debasish!
Tricky.
If I understand your data correctly, you show already normalised data in the excel file but not the raw absorbance data. ODs should be between 0.1 and 1.0 for reliable data interpretation.
Your positive control has nearly identical values like the other genes of interest. That's a bit puzzling and leads me to the following conlcusions:
A) all genes have the same effect (comment: unlikely)
B) The MTT assay is the wrong method for your question or not sensitive enough to detect small diferences (likely)
C) you should (dis)prove these initial data anyway with an additional method. I would suggest flow cytometry with staining for apoptosis. There are plenty of protocols for that in the web. I would also suggest to test multiple time points, e.g. 0, 4, 16, 24, 48 hours.
This gives an idea on the kinetics of action. It's more work but should also provide robust data and a better resolution of differences between treatments/genes. E.g. a 10% difference between gene effects cannot be detected as a significant difference in an MTT assay due to variance but would be significant via flow.
There are also other possibilities for MTTs to not work (photospectrometer not taken care of with calibrations or wrong wavelength, lamp bulb expired, MTT reagents didn't work properly,...).
My apologies that I cannot give better advise. Nevertheless good luck with the experiments!
@Alexander: Thank you very much for your valuable suggestions, we are now trying the Trypan Blur exclusion assays, but yes still it is a puzzle for me to how to get rid of it.
Alexander makes some great points,
One of the main problems that people often have with plate assays, as Alexander rightly says, is that they rely on absorbances that are too high (i.e. 3, which if you know the Beer Lambert law you will know this is not going to be reliable!) or too low (below 0.1). Also I would firstly suggest that you titrate the number of cells in your 96 well plate such that at the end point of the assay they are still in exponential growth rather than confluent becuase this will profoundly effect your toxicity- if you plate out 50,000 cells with a doubling time of 24 hours and your control cells are confluent after 2 days, you may kill half of your cells with your gene, but there may still be enough cells in the wells to reach confluence by the end point of the assay. I would also suggest that you do a "reverse" transfection where you mix the suspended cells with the transfection reagent and DNA in a tube before plating them out so all the cells should transfect at the same efficiency, and also keep some back to do a western or PCR to confirm expression. I have found that this gives much tighter results with plate assays.
The other thing is I am not sure whether you a measuring toxicicty/killing or inhibition of growth by MTT.
good luck anyway
PS try titrating the amount of your gene in your transfection mix- 25, 50 75 100% of your expression vector diluted with empty vector-if you get a good dose response it will just make your data more robust.
@Andrew: Thank you for your valuable suggestions. Now we are doing the titration studies with different conc of lipofectamine and DNA. Moreover by MTT we are trying to measure the growth inhibition effect actually but people often interpret it for cyto-toxicity effect , lets hope that we will get a good result for this experiments.. thanks again
Hi
I am also trying to do the same. I have doubt regarding transfection control?? how we have to normalize the transfection efficiency when we are transfecting a gene and checking the viability??
- Badges
- Science topic