I have the following issue:
I am currently working on the role of a miRNA (I will call this miRNA miR-X) on drug sensitivity in neuroblastoma (see figure pdf).
I found that this miRNA significantly induces differentiation in the neuroblastoma cell line that I am using.
In order to analyze whether the miRNA increases sensitivity to anticancer drugs, I had planned to use the AlamarBlue assay to measure cell viability. The alamarBlue assay is based on that living cells are metabolically active and are able to reduce the non-fluorescent dye resazurin to the strongly-fluorescent dye resorufin.
However, now I am speculating whether the alamarBlue assay is the “right” assay to measure sensitivity of cells to a certain drug in my case.
I found that transfection of cells with miR-X mimics significantly reduces cell viability (50% cell viability in miR-X transfected cells 72 hours after transfection- see figure pdf).
But I am now raising the question whether miR-X – transfected cells cells are really less viable as compared to control-transfected cells?
Does the alamarBlue assay rather indicate decreased proliferation and metabolic activity due to differentiation?
Can I really use the alamarBlue assay to evaluate whether cells are more sensitive to anticancer drugs?
Which assays may I use? I will analyze apoptosis by Flow cytometry and/ or western blot analysis. Do you have other ideas?
Some of the drugs that I am using induce mitotic catastrophe and not apoptosis. How may I measure drug sensitivity in this case?
Looking at your results tells me that there is no change in drug sensitivity with your miR transfection. Whatever effect you see is because of the transfection and not the drug. You could try cell counting using trypan blue and see if it helps. But if your experiment had no issues and you can absolutely believe your result, I don't see a point in not trusting the results you got. I do not think 18% decrease is significant enough to conclude drug resistance because of miR-X.
Dear Avinash,
thank you for your reply!
When using alamarBlue assay, I agree that there is no change in sensitivity. But I am raising the question whether this assay is the right way to analyze it in my case.
A recently published paper indicates "that inhibition of MYC is accompanied by accumulation of intracellular lipid droplets in tumor cells as a direct consequence of mitochondrial dysfunction" They found that "MYCN Inhibition Causes Respiratory Chain Impairment".
In my case (I have to further investigate this), it might be possibtle that the miRNA acts through targeting MYCN leading to differentiation.
As the alamarBlue assay is based on the activity of mitochondria, I think that this assay is not suitable in my case.
I found that Promega has several cytotoxicity assays. Does someone has experience?
Well perhaps you can have a closer look to other methods.
For determination of proliferation you can use trypan blue cell counting or MTT assay.
For determination of apoptosis you can use FACS analysis (e.g. Sytox, AnnexinV).
The combination of both should address exactly your issue.
best
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