Hi everyone, 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. The complete protocol can be obtained from http://aac.asm.org/cgi/content/short/48/5/1803. I also followed the improved protocol, which involves the washing steps to remove hemoglobin interference. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794876/)
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.