I want to analyze time dependently changes in gene expression of my CalB gene in Pichia pastoris. As for my house keeping gene I use ACT1.
For the rna isolation I use the kit from promega (SV total rna isolation, extraction with ethanol) and mini homogenizers. I incubate with Dnase I ( 2U/µL, thermo fisher) for 2 hours at 37°C and stop with the stop solution included in the kit.
I checked for rna integrity (agarose gel) and the concentration of total rna with Nanodrop.
Prior to my ddqPCR analysis I did a "Validation experiment" to check if CalB and ACT1 are quantified in a similar efficacy and to get the "dynamic input range". I always measure in triplicates. For this I analyzed the ddCT values of a sample in dilutions of 10ng to 0.01 ng. And there is the problem: the rna input amount is in a very low range dynamic. (around 0.02 ng gives me a slope of 30 for CalB and >24 for ACT1 but for my negative control around the same values as CalB !!
When I analyzed my reference sample (at time 0) again to check for reproducibility, i got dtCT values which differed around 4 CT values from the first measurement!
Any suggestions how I can get reproducible reliable ddCT measurements??
Thanks,
Chiara