I am doing a time course experiment. Treat cells with a drug for different times (0h, 2h, 8h, 18h, and 24h, respectively), and then harvest cells for RNA to check cell responses. So here we have 2 plans to do that.
1) Give drug at one time and harvest cells at different times.
2) Give drug at different times and harvest cells at one time.
In either way, cells will be treated for same hours. Which one do you think is the better one? And please let me know why. Thanks for any idea.
The first approach has the benefit that the treatment will be most consistent between the samples. The plates will contain the same number of cells (and will not have grown the extra 22 hours in one case) and the treatment can be done with a master mix that makes sure they are all treated the same. This would probably be best in that as long as your RNA preps work out, there shouldn't be much variability added at that stage. Further, there are lots of quality controls and internal controls in the RNA analysis.
The second approach would make the experiment easier in many ways as all of the RNA preps could be handled in parallel. However, you are adding some variables at the front end and those would concern me.
Difficult to make a suggestion, as your cells will grow between the time points, as you mention. If you can count the cells really very accurately and have a really standardized trypsination/seeding protocol, seeding them in the same number a certain defined time (22hrs as you are planning) before adding the drug and harvesting them all together might do the trick.
On the other hand, as your drug might have an effect on cell growth, I'd rather suggest to start all time points at the same time, as you have to normalize your data anyway.
For facilitating the RNA preps, you could add the phenol reagent to your cells, flash freeze them and still do the workup later to minimize variability (and possibly saving time).
We typically go with your second option. The cell density is carefully controlled in our experiments to ensure cells will be be similar at the harvest time. This means some pilot experiments before you do the round(s) where you harvest the RNA.
If you want to stick with option 1, you can reduce the number of controls by kind of blending both options. Harvest 0,2,8 at the same time. Then harvest another 0,18.24h at the same time on another day.
Really by plating the 2 and 8 hour at a slightly lower density (assuming your drug stops or slows growth), then harvesting all time points together is your best bet.
The first approach is better if you have a way to stop the reaction and freeze the cells to isolate RNA later. If you not, use the 2nd approach but having the same number of cells at each time point could be difficult if your cells grow faster (doubling time 24h or less).
Actually, I like Plan A. I can seed the same amount of cells, give the drug at the same time with same drug solution which is prepared in one tube. The only variable is " treatment time" which is comparable. For Plan B, too many variables. Have to prepare drug solution for several times, the start condition of cells is different. And my friend's lab likes that. I hesitate to follow.
But for Plan A, it will be midnight to collect samples at 8h or 18h...
ps: I think it is too difficult to make same cell density at the end of harvest time in plan B.
If time course is flexible, please use 4h instead of 18h to get some kind of linearity in the time course (0h, 2h, 4h, 8h, 24h), which will avoid you to come at midnight to collect the cells. We normally harvest the cells usig trizole reagent and store them -80C for RNA isolation at a later time.
No. Every time point is a representative point.
Thank you, everybody.
It has to be (2) for longer term cultures such as going from short times to 22h. This is only partly because the population will likely increase over that time period. I assume these are in monolayer.
You need to plate them all out at the same stage, and keep them in culture for exactly he same time.
in addition to growing, cells will secrete biochemicals that alter their properties and responses to external stimuli, as well as changing adhesion properties. Cells in cul=ture often have a master cell that can be identified. It pulsates so is thought be sending signals, in some way not yet understood, to the rest of the population.
Apart from growth increase, your population of cells at 22h is quite different from the same at earlier stages, not just in the size of the population, but in the way it responds. This has be shown for receptor numbers for example. With drugs you are sometimes looking at receptors, but more likely acceptors (similar to receptors, but les specific and mostly intracellular).
To have the level of accuracy and precision you require, you have to subject the cells to the same experience, except for the duration of being exposed to the drug.
Therefore to be able to do that, you really have to harvest at the end.
@Pauline Rose Mary Dobson, For the 2nd plan, when we add the drug to the cell culture at different time points, the cell growing conditions are different. For instance, when adding drug to cell culture for 8h treatment, cells already grow for 16h from the beginning. I think those may not comparable.
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