sir, i am new on R language and dealing with light-cycler qpcr data using HtqPCR package.everything goes all right till fold change. when i use t-test then following error occured. > qDE.ttest <- ttestCtData(sr.norm[, 1:2], groups = files$Treatment[1:2], calibrator = "Control") Error in t.test.default(x[, g1], x[, g2], alternative = alternative, paired = paired, : data are essentially constant when i select 4 samples then qDE.ttest <- ttestCtData(sr.norm[, 1:4], groups = files$Treatment[1:4], calibrator = "Control") Error in ttestCtData(sr.norm[, 1:4], groups = files$Treatment[1:4], calibrator = "Control") : Two factor levels required for 'groups' my parent file look like this File Treatment control.txt Control 30min.txt 30min 2hr.txt 2hr 4hr.txt 4hr 8hr.txt 8hr 12hr.txt 12hr 16hr.txt 16hr 24hr.txt 24hr 48hr.txt 48hr every sample have 26 features(13 replicate) so how i perform t- test on these data. please sir help me out. thanking you -- output of sessionInfo(): R version 2.15.1 (2012-06-22) Platform: i386-pc-mingw32/i386 (32-bit) locale: [1] LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252 LC_MONETARY=English_United States.1252 [4] LC_NUMERIC=C LC_TIME=English_United States.1252 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] HTqPCR_1.10.0 limma_3.12.1 RColorBrewer_1.0-5 Biobase_2.16.0 BiocGenerics_0.2.0 loaded via a namespace (and not attached): [1] affy_1.34.0 affyio_1.24.0 BiocInstaller_1.4.7 gdata_2.11.0 gplots_2.11.0 [6] gtools_2.7.0 preprocessCore_1.18.0 stats4_2.15.1 tools_2.15.1 zlibbioc_1.2.0 -- Sent via the guest posting facility at bioconductor.org.