Are you using the same concentration of input RNA for all of your samples or your input RNA concentration varies from sample to sample?

firstly, thank you for your answer.

I used same RNA concentration. it was 2ug of RNA.

for qPCR, I used 100ng cDNA per each sample.

I measured concentration of cDNA using NanoDrop. But some people said it can't measure cDNA concentration using NanoDrop.

I don’t know exactly what would be the reason for this. Unfortunately I am facing almost the same issue.

It might be due to sample to sample variation. That’s the reason you use normalization control.

Can you tell me what are your Ct values? Is the Ct value for your control of both gene of interest and reference gene in the same sample differs from other sample?

Sure. the following are my Ct values for control samples per each attempt.

trial#1 Ct (GAPDH): 23.134 / Ct(Target gene):29.261

trial#2 Ct (GAPDH): 23.783 / Ct(Target gene):29.252

trial#3 Ct (GAPDH): 31.140 / Ct(Target gene):30.435

I think the problem was cDNA loaded amount.

I thought whatever I loaded amount little differently, it doesn't matter because the difference between target gene and house keeping gene is important, before.

but now I think the loaded cDNA amount may effect to the difference between Ct of genes.

so now I am gonna try to load same amount per each attempts.

Yes using different concentration of cDNA affects Ct values. Try making serial dilution of your cDNA and then do PCR. there should be proportional increase in Ct value with each dilution.

OK, I am going to try it.

thank you very much.