I'm currently working on my final year project, which is to screen for plants with antimalarial activity. I'm using the fluorescence method which involved the use of SYBR green I dye to determine the parasitemia level of the parasite culture treated with different plant extracts. Hence, the effectiveness of the extracts are evaluated by determining their respective IC50 (inhibitory concentration at 50%) values.
The complete protocol can be obtained from http://aac.asm.org/c...hort/48/5/1803. I also followed the improved protocol, which involves the washing steps to remove hemoglobin interference (http://www.ncbi.nlm....les/PMC2794876/), but it din't help.
I'm facing a problem in this assay. The results for my positive (infected erythrocytes) and negative (non-infected erythrocytes) controls are contradictory, their relative fluorescence units(RFU) appears to be very close to each other. In a few tests, the RFU of negative controls are even higher than the positive controls. I have to get this right before I could start the screening.
I would very much appreciate your generous help if any of you could advise me on how to optimize the assay. Thank you.
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Testing antimalarial activity in plants using SYBR green
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