I am searching for MECP2 gene duplication ( X linked ) in males with mental retardation using SYBR green real time PCR relative quantitation method . We are comparing the gene copy number in male patients versus female controls and we have a reference housekeeping gene . At optimization as a first step , we used normal males and female controls .When using the 2^ (-dd CT ) method in RQ calculation , it was supposed that , females will be double that of normal males due to presence of 2 X chromosomes in the former and only 1 in the later . The results came to be crossed or bizzare and also the CTs of males are less than that of females which is unlogic , meaning that the male amplification is earlier . The ST curve efficiency is 105-107% .

What do you think may be the error ? Shall we use Pfaffl method instead or Is ST curve efficiency more than 105 % may be the problem ?

Thanks in advance!