Hi to all! I've to perform a time series analysis with timecourse package, but I've got plotting problems and some doubt on experimental design. I've 2 cell lines (first as parenthal, second as transfectant for a specific gene), each with 2 biological replicates and 4 time points, for a total of 16 samples. Main goal is to find genes with different temporal behaviour between parenthal vs. transfectant. Everything seems to work properly until plotting function, that generate this error: > plotProfile(MB.2D,type="b", gnames= row.names(Data)) Error in rep.pch[1:nrow(con[!is.na(con[, 1]), ]), 1] <- pch[1:nrow(con[!is.na(con[, : subscript out of bounds and plot is empty but title reports probe name, statistics and rank. Here how I set mb.long function: # perform indipendent two-samples analysis > MB.2D <- mb.long(Data, method = "2", times = 4, reps = size, > condition.grp = condition, rep.grp = groups, time.grp = time_exp) And here results of mb.long (that looks right): # size matrix > head(MB.2D$size) [,1] [,2] [1,] 2 2 [2,] 2 2 [3,] 2 2 [4,] 2 2 [5,] 2 2 [6,] 2 2 # condition > MB.2D$con.group [1] "Parenthal" "Parenthal" "Parenthal" "Parenthal" "Parenthal" [6] "Parenthal" "Parenthal" "Parenthal" "Transfectant" "Transfectant" [11] "Transfectant" "Transfectant" "Transfectant" "Transfectant" "Transfectant" [16] "Transfectant" # replicates > MB.2D$rep.group [1] "t0" "t0" "t7" "t7" "t14" "t14" "t21" "t21" "t0" "t0" "t7" "t7" [13] "t14" "t14" "t21" "t21" # time > MB.2D$time.group [1] 1 1 2 2 3 3 4 4 1 1 2 2 3 3 4 4 Input Data is a txt file as matrix of log2 expression data from previous filtering steps with limma, imported with: Data <- read.table("./Input_file.txt", header = TRUE, row.names = 1) Is it a plotting problem, or could be also a wrong experimental design that couses subsequent error? Further question, for each probe I get a Hotelling statistics value: which is the "significant" threshold (I get values from 2330 to 0.006)? sorry but I'm more confidential with p-value.. Any support is really appreciated, thanks in advance, Andrea Dr. Andrea Grilli andrea.grilli at ior.it phone 051/63.66.756 Laboratory of Experimental Oncology Rizzoli Orthopaedic Institute Codivilla Putti Research Center via di Barbiano 1/10 40136 - Bologna - Italy