Hi,
My team was trying to profile bacterial phylum composition in food sample using qPCR. We used 16s universal primers and primers specific for certain phylum such as Firmicutes and Proteobacteria. By using this approach we expected to be able to calculate how much the ratio of a certain phylum compared to the total number of bacteria. We used E. coli as standard for amplification with universal primers and other pure culture for the other phylum-specific primers (e.g. Lactobacillus for firmicutes). When the result came out, we found that in some samples, the copy number using phylum specific primer was higher than amplification of the same sample with universal primer. Logically this should not be possible since that implies we have more firmicutes for example than the total number of bacteria in that sample. What would be the explanation behind this? Is qPCR technically feasible for the analysis that we were planning to do?
Different primers = different assay. You cannot compare Ct values between different assays directly. Use an external standard to calibrate your measurements.
Two possibilities:
1. Check that the efficiency of both qPCRs are the same or very similar. This can be done with a calibration curve and the following formula (E = 10^(-1/slope).
It should be for both qPCR around 100%. If not, try changing some parameters.
2. The universal primers are not so universal and usually amplify some genus better than others. Try another universal 16S primers.
Good luck!
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