Hello,

I am performing RTqPCR, analyzing several bacterial genes for their expression.

When I check the efficiency of the primers using gDNA serial dilutions, the efficiencies look ok, between 90 and 100 %. When then I check the efficiency using serial dilutions of my cDNA I have very high efficiencies (> 130%).

To extract the RNA I use qiagen kit and to obtain cDNA I use Promega kit (AMV Reverse Transcriptase).

I do not have primer-dimers.

I should also say that I work with obligate intracellular bacteria, therefore I always carry host cell RNA with me.

I am running out of ideas, so any suggestion is appreciated!

Thank you!