I would like to fraw your kind attentions towards my problem which i am facing while standardization of multiplex real time assay.
I am planning for comparative gene expression analysis in cancer where i wanna look for overexpression of a target gene in disease condition. I want to standardize Multiplex real time pCR assay by employing target as well as house keeping gene in the same tube.
I am getting successful amplification of both the genes when i am performing it in separate tubes, but as i go for multiplexing both the genes in tha same tube i am failing to get amplification plots for both the genes.
The probes are labelled with FAM and Hex with TAMRA as a Quenchor. I have tried initially by keeping the target primer and probe conc same and varied the conc of house keeping gene's primer and probe but no success was achieved.
SO PLEASE SUGGEST AND GIVE ME YR INPUTS AT THE EARLIEST.
Need yr suggestions for multiplex realtime PCR standardization!