TaqMan Gene Expression Assays - Low expression/ Ct values (Jul/16/2010 )
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!!
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
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.