Dear Professor Burzynski

Thanks for your worth able comment, in replay to your question I must say, YES, I did conventional PCR separately for each gene and they worked by a Tm around 56 however not worked for cox2 when I run for Multiplex.

 I corect the above  writing  as Ta is around  56 for two genes.

Dear Mohsen, have you tried real-time PCR or standar-PCR separately? Moreover, Sybrgreen did not allow you to specifically tag a couple of primer so, just to understand, are you expecting more than one amplification curves? If you don't apply a Melting Curve analysis after the amplification, I am not sure you will be able to distinguish the two amplification curves. I am working with similar things, but I use Taqman probes.

Dear Dr Mohsen,

It seems to me that the problem is due to the topology of the different targets being used in your qPCR. in addition to the template topology, the length of amplicon also results in biased amplification, smaller amplicons are preferred during co-amplification. We have also seen this in our works. Please go through these excellent works which I think can help you a lot to understand the issue:

Chen J, Kadlubar FF, Chen JZ. DNA supercoiling suppresses realtime

PCR: a new approach to the quantification of mitochondrial

DNA damage and repair. Nucleic Acids Res. 2007;35:1377–88.

Laghi L, Randolph AE, Malesci A, Boland CR. Constraints

imposed by supercoiling on in vitro amplification of polyomavirus

DNA. J Gen Virol. 2004;85:3383–8

Lin CH, Chen YC, Pan TM. Quantification bias caused by

plasmid DNA conformation in quantitative real-time PCR assay.

PLoS One. 2011;6(12):e29101.

 SD

Dear Professor Burzynski , Dr, Cavallero and Dr, Datta 

Thanks so much as Participate in discussion,  each of you mentioned an important aspect of the problem to me while the articles that Dr, Datta introduced were wonderful to open the puzzle for me so I propose reviewing of them to other friends as well. 

Best regards

Mohsen

I completely agree with Prof Arthur. It does affects the PCR efficiency, and if you linearize the template before PCR, one can have even better efficiency in multiplex PCR also. there was one paper, most probably in Nucleic Acids Res or PLOS One, which showed that in multiplex Real time PCR assays, longer targets are less efficiently amplified as compared to the smaller amplicons. If i get the paper, I will share.

Meanwhile, we have recently published an article on pre-PCR template linearization, to enhance PCR efficiency, which you can see, if applicable to your work.

https://www.ncbi.nlm.nih.gov/pubmed/26993322

Thank you,

SD

Thank you Dr Datta 

The problem is the same with me as my large amplicon is around 190 bp for Cox2

while the shorter amplicon is around 58 bp for ITS gene.would you please send me the 

target adress  of the paper in PLOSE one.

Sure Dr Mohsen,

I am trying to find out that very paper, and shall send you if I get.

Best wishes,

SD

Dr. Mohsen;

One of the probability that you have suggested may be one of the reasons. Before trying to put one's finger on the most probable answer, I would suggest that instead of running the PCR simultaneously, just try running it seperately and see what you get. My prediction is competitive compatibility.

Dear professor  Ghost

Many thanks for your comments.

Best ...

Mohsen