Hello everyone,
I conducted a series of experiments in which I compared the efficacy of an anticancer agent with different viability tests. I repeated five wells for all drug doses in each test. As a result of the experiments I conducted under the same conditions, the standard deviations of my MTT test were quite low, while the standard deviations of my SRB and DNA fluorescence tests were relatively high.
What is the reason for this situation? For example, it occurred to me that the SRB test involves too many washing steps. Or could it be related to the sensitivity of the device I'm reading the plates?
I am attaching the top part of the graph for you to see.
Thank you so much.
Maybe I should simply tell you that
The fact that the standard deviation is low in one place and high in another has nothing to do with statistics. It has to do with your own knowledge and how to measure data.
so simple
When the standard deviation is small, it means that the data is stacked like close friends. Why? We do not know. That is, we do not know as a statistics.
And
When the standard deviation is large, it means that the data are angry with each other. Why? We still do not know. This is your data.
Dear Abolfazl Ghoodjani ,
I understand your way of thinking from a statistical perspective, but one way or another there must be a reason for this situation. As I said, it may even be related to the sensitivity of the device I'm measuring. In this case, I may choose not to trust my results or change my conditions, I think.
Thank you for your answer!
Hello Gülşah Torkay
There are many points to be considered when you perform SRB assay, the washing step is one of them.
1. The seeding density should be such that when the plate is read on the final day cells should be around 70-80% confluent.
2. Ensure even distribution of cells at the bottom of the plate. The best way would be to add cell suspension directly to the bottom of the well. Do not touch the sides of the well.
3. During cell fixation gently add TCA to each well directly to the medium supernatant. Do not mix as it would cause cells to detach from the bottom of the well.
4. While washing the plate, submerge the plate in a tub with slow running tap water. Cell monolayer can detach if water is forced into the wells. The plate must dry completely before proceeding to the next step.
5. While adding SRB to each well ensure that the solution is in direct contact with the bottom of the well with no air bubbles.
6. After SRB incubation rinse the plate quickly with acetic acid to remove unbound dye. An important point to be noted is that non-homogeneous washes is the major source of variation in this assay. So, washes must be done quickly and homogeneously across the plate. You need to visually check that acetic acid has been dispensed in all the wells and that there are no air bubbles which could prevent the washing of the wells. You can dispense the solution into the wells from the side walls of the well.
7. Once you add Tris base solution to each well do shake the plate on the plate shaker for 10 mins to solubilize the protein bound dye.
If you take into consideration all these points this problem may not arise.
I hope this will help.
Good Luck.
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