Dear Heidi, I am trying to tailor HTqPCR.R such that it can readCtdata into an qPCRSet. I have manually constructed my dataset with 162 gene features, and two technical replicates, generated from 96-well plate. I have formatted the data (162:3) as such col.names=c(feature=1, type=2, Ct=3: > data<-read.delim("lv_TechRep.1.txt") > data featureNames featureType Ct 1 lox22 untreated.0d 22.28 2 actin22 untreated.0d 20.30 3 actin22 control.1d 20.91 4 actin22 control.3d 21.08 5 actin22 control.7d 21.00 6 actin22 control.14d 21.32 7 lox22 control.1d 23.65 8 lox22 control.3d 24.77 9 lox22 control.7d 23.99 10 lox22 control.14d 24.47 11 actin22 treated.1d 21.36 12 actin22 treated.3d 21.22 13 actin22 treated.7d 21.79 14 lox22 treated.14d 22.07 15 lox22 treated.1d 22.13 16 lox22 treated.3d 21.99 17 lox22 treated.7d 24.17 18 lox22 treated.14d 24.17 19 lox23 untreated.0d 24.86 20 actin23 untreated.0d 23.22 21 actin23 control.1d 21.09 22 actin23 control.3d 21.81 23 actin23 control.7d 21.52 24 actin23 control.14d 21.33 25 lox23 control.1d 25.87 26 lox23 control.3d 26.78 27 lox23 control.7d 26.38 28 lox23 control.14d 27.92 29 actin23 treated.1d 21.47 30 actin23 treated.3d 21.35 31 actin23 treated.7d 21.68 32 lox23 treated.14d 22.04 33 lox23 treated.1d 25.77 34 lox23 treated.3d 26.03 35 lox23 treated.7d 27.31 36 lox23 treated.14d 28.11 37 lox28 untreated.0d 23.85 38 actin28 untreated.0d 19.96 39 actin28 control.1d 20.35 40 actin28 control.3d 20.70 41 actin28 control.7d 21.14 42 actin28 control.14d 21.00 43 lox28 control.1d 19.96 44 lox28 control.3d 23.85 45 lox28 control.7d 25.15 46 lox28 control.14d 25.91 .... 162.... I've tried to read in the data using raw<-readCtData(c("lv_TechRep.1","lv_TechRep.2"), path=path, n.features=162, column.info=(c(flag=NULL, feature=1, type=2, Ct=3, position=NULL)), format="plain", header=T, n.data=1)) However, I get error messages: Error in readCtData(c("lv_TechRep.1.txt", "lv_TechRep.2.txt"), path = NULL, : argument 7 matches multiple formal arguments Can I not list the TechReps.n in readCtData prior to combineTechReps? Does HTqPCR not read my file because of file.dim()? I've tried to use ReadqPCR, qPCRNorm and ddCt functions but cannot read CtData into an eSet. I've also tried to read in read.delim and data.frame, then convert this to a matrix(). However, I continue to get error messages when trying to form my rownames: rownames(temp)<-paste("gene", 1:162) Error in `rownames<-`(`*tmp*`, value = c("gene 1", "gene 2", "gene 3", : length of 'dimnames' [1] not equal to array extent Any suggestions on the data formatting (both tech.reps are identical except for Ct values) or how to write in the data to make an qPCRSet. I've seen the previous postings, however, this was not that helpful for my situation. Best Regards, Franklin -- output of sessionInfo(): > sessionInfo() R version 2.15.1 (2012-06-22) Platform: x86_64-pc-mingw32/x64 (64-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] ddCt_1.12.0 lattice_0.20-13 xtable_1.7-0 NormqPCR_1.4.0 ReadqPCR_1.4.0 affy_1.36.1 BiocInstaller_1.8.3 [8] HTqPCR_1.12.0 limma_3.14.0 RColorBrewer_1.0-5 Biobase_2.18.0 BiocGenerics_0.4.0 loaded via a namespace (and not attached): [1] affyio_1.26.0 gdata_2.12.0 gplots_2.11.0 grid_2.15.1 gtools_2.7.0 preprocessCore_1.20.0 [7] stats4_2.15.1 tools_2.15.1 zlibbioc_1.4.0 -- Sent via the guest posting facility at bioconductor.org.