Real time PCR of low expressing genes - Late tumor Ct (Feb/11/2007 )
I am trying to do a real time PCR (relative quantitation) in human breast carcinomas to find a specific interleukin expression. I know that this gene is up-regulated in tumors and is low expressing in normal breast tissues. So, i'm doing my tests... The Ct of my tumor samples are late (27-31) and GAPDH (22-26). Tomorrow I will do it in normal breast samples...but, if the gene is considered low expressed and in the tumors it is already low expressing (with late Ct),...if the normal breast samples don't amplificate? How can I analize my results? I can't do absolute quantitation (I do not have money).
Is it possible to do a real time PCR (relative quantitation) with a normal (control) without expression?
I think that if I concentrate my samples I will change just few Cts... will not resolve my problems.
Don't be so stress.
When you load your real time PCR, put the sample control (negative) in the same time as yours samples.
The normal breast sample is a good point, but I think it 's better if they are on the same plate of the others.
GAPDH is also a control , It show you that you have the same amount of material in each sample because it will never change the Ct (not so much).