What is the significant fold increase for delta delta ct analysis for a prokaryotic gene. Is it above 2 fold? If so, have any one have got above two fold increase in any prokaryotic gene?
Iam doing this assay for efflux genes, but iam not getting above 2 fold. even though the strains have high efflux activity.
Given your assay is working well, then I would think of the possibility that the efflux is controlled at some non-transcriptional level (protein synthesis, degradation, modification, location etc).
You are making two assumptions (one biological and one technical).
Biological, are you sure that these efflux proteins are regulated at the transcriptional level? You may not be sampling at the correct timepoint or it's not a transcriptionally regulated system.
Technical, what are your primer efficiencies? If your primers are not 95-105% efficient, then you need to change your reaction conditions. Also, if your primers have different efficiencies from each other, you will need you account for this difference with the Pfaffl correction.
Thank you all for your valuable suggestion. The primers are good. Mostly biological rather than technical. Iam still working on it and i will let you know about it.
thanks again.
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