I personally like using REST2009 for RT-qPCR analysis as it is really easy and gives you great graphics and quantification. It should have no problem handling your treatment and control group - I hope you have Cts of reference gene quantification for all samples?

http://www.gene-quantification.com/rest-2009.html

Adding as attachement because ResearchGate doesn't permit pasting of replies in IE 11, and I'm not going to retype my reply.

I perfomed my RT-PCR using two samples of cDNA (unstimulated and stimulated). For every samples I evaluated the gene expression of the endogenous control (beta-actin) and human beta defensin 2 or 3.

For example, for the beta defensin 2, I obtained these deltaCT:

1) Unstimulated: beta-actin (27.096) + beta defensin 2 (no deltaCT);

2) Stimulated: beta-actin (24.679) + beta defensin 2 (37.693);

So, if i try to calculate the fold of increase, I will obtain numbers too high.

With the CT values being so high for Beta Defensin, I doubt you can interpret the data in any case. Any CT over 35 is close to nothing. Unless you can validate the linearity of your assay above a CT of 37, there's nothing you can say about the Beta Defensin expression levels from this data.

You may need to try using more cDNA, or using different primers, or optimizing the assay to lower the CT for Beta Defensin.

Hi Giacomo,

From my point of view try to use more than reference gene (at least three) and see what the results will be?

Good luck,

Dalia