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$Treatment[1:4], calibrator = "Control") : error:: Two factor levels required for 'groups' my parent file look like this File Treatment control.txt Control 30min.txt 30min 2hr.txt 2hr 4hr.txt 4hr 8hr.txt 8hr 12hr.txt 12hr 16hr.txt 16hr 24hr.txt 24hr 48hr.txt 48hr if i change name as following File Treatment cnt.txt Control 30min.txt Treatment 2hr.txt Treatment 4hr.txt Treatment 8hr.txt Treatment 12hr.txt Treatment 16hr.txt Treatment 24hr.txt Treatment 48hr.txt Treatment then it works qDE.ttest <- ttestCtData(sr.norm[, 1:4], groups = files$Treatment[1:4], calibrator = "Control") what to do bcz my samples have no replicate and groups consist of only 2 sample not 4 as in example file. every sample have 26 features(13 replicate) so how i perform t- test on these data. please sir help me out. thanking you. [[alternative HTML version deleted]]