Hi, this is my first time on this forum. I came to stop in my experiment with Real Time due to this problem. i am observing two genes of interest in a sample (multiplex). On my first run, my readings for my Sample: Gene A: 9.8851 9.9885 9.835
Gene B : 31.9156
31.7019 31.9177

well, its a good result as the standard deviation is less then 0.2

Nevertheless, on my second round of Real Time of the same assay
my result for the same sample Gene A : 13.7914 13.967 13.5537
Gene B : 31.967
31.5581 31.9262

You see.. for the Gene B value (CT) remain the same(reproducible) but Gene A which is a stable endogenous control varies.

Well, can you tell me why this happen? What factors affect the Ct values?
to note that my cDNA is of the same tube.

As I can see here... the Ct value for Sample B is remaining constant as there is very less or negligible DNA in it. Sample A is giving this variation as there is huge amount of template that is going in your wells which might not be optimum for the concetnration of primers dNTPs and Mg ions that you might be using. So this is mere problem in the high concentration of your template. If you dilute your sample in Sample A and then do it twice or thrice you should not get this varuation in Ct value!!!!