Hi Hiedi, My name is Stefany I'm starting to learn R very recently so my expertise is limited. I've been reading your manual an some post from previous questions regarding how to re-format data to process with HTqPCR package. My data come from an Applied biosystems machine. I have to samples, two conditions, 5 target genes and a control gene. But each sample has been measure over a time course (5 time points in total). Each sample has been tested for each gene in triplicate. In total I have 2 conditions*5 time points*6 genes* 3 . This doesn't fit the default parameters for HtqPCR. I read  from someone else post that you mention two ways to go around this. One is to create a new qPCRset and other is using the function readCtLayout. My questions are, should I calculate first the average of each replicate an with these values create a new qPCR set (so I'll have a single sample per each gene)?, can I also have an arbitrary number of columnsxrows (one that doesn't fit the 384 rows?) so the structure of the matrix fits my samples/genes? I'm a bit confused, do I need to have all the headers you have mention in previous posts (Plate Pos Flag Sample Detector Task Ct Delta Ct Avg Delta?). I'm trying to create the matrix but I'm confused on how to format my data.

Also is readCtlayout changed by changeCtlayout? 

I've been thinking if maybe my experiment design is not suitable for this type of analysis. My aim is to get the fold expression, significance in variances and a differential expression analysis on my data... I want to normalise my data using the deltadeltaCT method, but reading your manual, there are other type of interesting analysis I could implement with my data. The manual is very well explain, my main problem is to format my data the right way. So any help, advice is more than welcome please.

Stefany