Dear, I'm having a problem with the ttestCtData function from the HTqPCR package, which I use to analyze my BioMark Fluidigm data. In most cases there is no problem: > qDE.ttest.Nanog <- ttestCtData(q.norm[,c(1:9)], groups = conditions[c(1:9)], calibrator = "R-L_KD", stringent = FALSE) > head(qDE.ttest.Nanog, n=2) genes feature.pos t.test p.value adj.p.value ddCt FC meanCalibrator meanTarget categoryCalibrator categoryTarget 40 Nanog feature30 -16.290155 2.477252e-05 0.002204754 1.2277206 0.4269915 15.50882 16.73654 OK OK 84 Zcchc12 feature71 6.271628 4.370851e-04 0.019450286 -0.5513761 1.4654829 16.03707 15.48569 OK OK However sometimes I get this error > write.table(qDE.ttest.Nanog, file = "/Users/ruben/Dropbox/Data/qPCR/ results/BioMark/Fluidigm4/ND2_fluid4/Ttest/ND2_ttest_Nanog.txt") > qDE.ttest.Rest <- ttestCtData(q.norm[,c(1:5,10:13)], groups = conditions[c(1:5,10:13)], calibrator = "R-L_KD", stringent = FALSE) Error in t.test.default(x[, g1], x[, g2], alternative = alternative, paired = paired, : data are essentially constant However when I change the normalization from quantile normalization (q.norm) to for example norm.rankinvariant (nr.norm) this error doesn't occur anymore. All of the samples are different, I compare 4 biological target replicates to 4 from the control. Could it be due to the quantile normalization? And would it be ok if I used the norm.rankinvariant normalization if I encounter this error? Best regards, Ruben [[alternative HTML version deleted]]