Currently, I am working on an aggressive breast cancer cell line. I intend to restore and enhance the expression of tumor suppressor genes by siRNAs against the DNMT gene and induce apoptosis after treating the cells with siRNAs and subsequently exposed to a chemotherapy drug. I performed the MTT and Annexin V/PI apoptotic assays and I have got weird results.
In MTT assay the conc. of chemo drug with no siRNA was ranged from 100nM to 3200nM and the incubation time was 12 hours. In that experiment, the IC50 was 270nM. But, In cells treated with siRNA and the drug, even the highest conc. of the drug, i.e. 3200nM, did not induce cell death (the ODs were the same for each conc.). To clarify, I reverse transfected the cells with siRNA using RNAimax lipofectamine by Thermo fisher protocol (12 hours incubation time). subsequently, replaced the medium with the serially diluted conc. of the drug in RPMI containing FBS. after 12 hours of incubation, I have proceeded with MTT assay.
In the annexin V/PI experiment, I reverse transfected the cell only with the siRNA. the percentage of the cells in the Q4 quadrant was 90%!! As I understand, the siRNA against DNMT is supposed to induced apoptosis. but the results are showing a completely different outcome.
I have done the same procedure on CMML cells and the results were showing a considerable induction of apoptosis. the only difference was the transfection time, 19 hours against 12 hours.
Any ideas?
It may be that the transfection has failed. Have you tested the transfection efficiency of siRNA in the target cell line?
Linna Green That may be the case. But, if the transfection has failed, why is the chemo drug not working after replacing the transfection complex with the concentration gradient of the chemo drug in the MTT assay. The IC50 of the chemo drug is about 200nM, and the highest concentration of the drug I used was 3200nM after the transfection process. The viability of the cells in the 3200nM treated drug was even higher than the IC50 of the cells only treated with the drug and no transfection. I searched online, and I think maybe I need to wait 24 hours after transfection and then further the processes. If that is the case, there is another problem. My cell line is aggressive, and the cells die if I wait 48 hours after seeding as they reach 100% confluency. I even reduced the serum dependency down to 5%. I am really desperate because of my cell line doubling time.
Linna Green and no, I have not tested the transfection efficiency. Because of the sanctions against Iran, I am short in recourses and materials, including lipofectamine RNAimax. I thought if I perform my experiments by the general information in Thermo fisher protocol, maybe it is more efficient. But it seems I was wrong. Do you have any suggestions on how I could optimize the process by using low amounts of lipofectamine?
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