Inconsistency of the cells~~ - (Jan/21/2007 )
I perform a few assays on my transfected cell lines but the cells seem lacking consistency~~
the readings differ a lot from time to time
I am sure it is the problem of the cells but not the method used, because the reading is similar when I reapeated with the same patch of samples.
What is the probable causes of the inconsistency and how can I prevent it?
Do you mean the levels of expression vary among experiments done at different time, or different wells of samples in the same experiment? For the first case, it is common. If you can limit the variation within 30% for separated experiments, you are a pro. If you are dealing with the second case, that is mainly your technique. Pipetting error, the number of cells as well as how well cells distribute in the wells make the difference. You just have to keep everything as consistant as possible. Sometimes, increase the number of wells can reduce the variability within the same group.
We used to repeat the same experiments and look at the change in % within each group with regard to the controls and treated ones.
Because if u r going to compare 2 dif. sets of samples (transfected on different days, there will b inconsistencies).
let me know if u understood.
To genehunter-1, I think I am the first case. I tried to reduce the error, but not very successful.
To scolix, I have repeated the experiment many many times but the trend was not consistent at all~~
I think it's the problem of cell growth, they might be at different stage everytime when I collected the cells
May b I was not clear.
One has to compare control and treated groups within each experiment ( u have to do atleast 3 different experiments) as everytime the cells grow differently and transfection works differently everytime. So its difficult to compare one experiment to another one directly.
One would have to calculate the changes (may b in percentage) within an experiment and then compare it to another set of experiments ( where u have repeated the very same thing but on a different day).