Dear all, I have a data set that consists of 36 qPCR plates: 3- cond1 2-cond 2 1-cond3 when I make the normalization using the endogenous gene I get the following message: Calculating deltaCt values > > Using control gene(s): U6 > > Card 1: Mean=33.62 Stdev=NA > > Card 2: Mean=32.65 Stdev=NA So after seing the normalized data, I saw that the normalization is just making the difference between the Ct of the endogenous gene and the Ct of the target gene. Shouldn't the normalization take into account the variance of the Ct value of the endogenous gene across all samples and conditions in order to normalize and create comparable expression values between conditions? With kind regrads, Andreia -- -------------------------------------------- Andreia J. Amaral Unidade de Imunologia Clínica Instituto de Medicina Molecular Universidade de Lisboa email: andreiaamaral@fm.ul.pt andreia.fonseca@gmail.com [[alternative HTML version deleted]]

Dear Andreia your question is perhaps too vague to attract people who may want to answer it. Can you provide a reproducible example and the output of sessionInfo()? Best wishes Wolfgang On Sep/10/10 8:55 AM, Andreia Fonseca wrote: > Dear all, > > I have a data set that consists of 36 qPCR plates: > 3- cond1 > 2-cond 2 > 1-cond3 > > when I make the normalization using the endogenous gene I get the following > message: > > Calculating deltaCt values > >>> Using control gene(s): U6 >>> Card 1: Mean=33.62 Stdev=NA >>> Card 2: Mean=32.65 Stdev=NA > > So after seing the normalized data, I saw that the normalization is just > making the difference between the Ct of the endogenous gene and the Ct of > the target gene. Shouldn't the normalization take into account the variance > of the Ct value of the endogenous gene across all samples and conditions in > order to normalize and create comparable expression values between > conditions? > With kind regrads, > Andreia > > > > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor -- Wolfgang Huber EMBL http://www.embl.de/research/units/genome_biology/huber