2 replies to this topic

[George250]

Hey guys

I am working with TaqMan Gene Expression assays. Could someone please tell me what is the max Ct values which are accepted in QPCR? I am working with low cell numbers (50-100) and one of the genes I am looking should be expressed at very low levels. Would you consider Ct values above 35 normal when low gene expression is expected?

ps. I am using rotor gene 6000

Any information would be much appreciated!!

Thank you very much!!

[brenda anne]

hi george. i’d say that above cycle 35 the variability will be too high and the quantification unreliable    -you can check nature protocols 2006 1(3):1576
or it could be simply considered noise -nucleic acids research doi:10.1093/nar/gkn725
i hope this helps  

[ElHo]

I´d say it depends on the gene and how accurate your quantification has to be. Try doing a dilution curve to get an idea of the assay limits. Make replicates during one run and different runs to check for inter- and intra-assay variability and see if you get consistent results for higher Ct values.