I've got a basic question in qRT-PCR. I am new to that but I am sure that many of you faced this problem before.
I am just simply need to do a qPCR but my samples' RNA concentration is very low (ranging from 2.7-1.6ng/uL).
As I calculated, the maximum amount of cDNA in a 10uL qPCR reaction would be 0.549ng.
Before measuring the genes of interest, I first run the housekeeping gene Gapdh to see what is the Ct value in order to decide should I go ahead for other genes. I've got 23-28 Ct among the samples. So, it's already close to 30.
I am sure that the expression level of my genes of interest are much lower than Gapdh (10-15 Ct behind).
Now, I think that I could not get reliable or even cannot get a Ct value of those genes.
So, is there any method that I could detect those genes?
Isn't it concentrating the RNA is the only way?