Need yr suggestions for multiplex realtime PCR standardization!

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Firoz Ahmad
Firoz Ahmad's picture
Need yr suggestions for multiplex realtime PCR standardization!

Hello Everyone!
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.

Tony Rook
Tony Rook's picture
Firoz Ahmad:

Firoz Ahmad:

Here is a link to one of the best quantitative real time PCR sites available - www.Gene-Quantification.info

Here is the introduction summary presented on the site:

The Gene Quantification page describes and summarizes all technical aspects involved in quantitative gene expression analysis using real-time qPCR & qRT-PCR. It presents a lot of applications, chemistries, methods, algorithms, cyclers, kits, dyes, and services involved. Comercial and academic institutions can present their qPCR tools right here.

This site is maintained and edited by one of the premier researchers in the field of quantitative RT-PCR, Dr. Michael Pfaffl.

Here is the direct link to the PCR optimization page from this site:

http://optimisation.gene-quantification.info/

Good Luck!

Firoz Ahmad
Firoz Ahmad's picture
Dear sir,

Dear sir,
Thanks for yur suggestions and important information.
With regards
Firoz