I want to detect gene expression by multiplex PCR. The multiplex PCR reaction works fine on genomic DNA, but on a cDNA template the same oligo mix seems not to work as nicely, although specific bands are amplified by singleplex (i.e. regular) PCR reactions without problem. I tried different concentrations of oligos and cDNA template without great success. Adding magnesium ions or DMSO isn't of any help apparently. Does anoyone have experience with multiplex RT-PCT and can provide his/her expertise on this?
You may contact Karo Wagner in my lab ([email protected]).
Regards,
Peter
Does your PCR on cDNA template poisonned by the RT mixture ?
your primers work well on genomic DNA because this is a purified template whereas the cDNA is not...
-> try to dilute more your RT reaction in your PCR: until 1/50, 1/100
-> try to purify cDNA by phenol/chloroform, precipitation...
your primers could also recognized several other sequences in the RT reaction which are absent in genomic DNA and this interfer with the efficiency of the specific PCR. Is there any possibility of alternative splicing in your cDNA targets ?
-> try to design other primers
Hab Dank @Peter. Ich hoffe, dir geht es gut? LG nach Zürich
Thanks @Eric. This is not the issue though, since the singleplex PCR ractions work fine on cDNA. Just the multiplexing on cDNA seems problematic. I already tried different dilution and the primers bind in exon regions.
you may need adjustment of you tm of your primers eg.lower your tm >>>the main cause of multiplex PCR failure
@Ibtesam Primers work perfectly fine at the chosen Tm on gDNA in multi- and singleplexes as well as on cDNA in singleplexes.
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