Hi, There has been quite a bit of discussion on this listserve about dealing with technical replicates (in sets of arrays where the gene layout is the same across the experiment). We have just started analysis of a Stanford published data set where different print runs have a different set of genes replicated (ie different layouts). For example on Chip 1 (treatment) we may have clones A,B,X,Y and on Chip 2 (treatment) we may have A,A,B,X,X, Y and on Chip 3 (control) we may have A,A,B,X,Y and Chip 4 (control) we may have A, B, X, Y. For simplicity, I have only two chips for each condition in this example. Below I have the 3 alternatives for analysis and the pros and cons as we see them at the moment. I would really appreciate any comments anyone may have on our thinking! (1) Averaging the technical replicates within an array Pro's: (a) they are the same DNA sequence on the array so an average is meaningful (b) it would be easier for downstream analyses to have a single representation on a chip, Con's: you will be doing a linear model on averaged data and non-averaged data on each chip so the spots are measured with different precision. (2) Analysing all combinations eg for clone A: Chip 1 has A1, Chip 2 has A2, A3, Chip 3 has A4, A5, Chip 4 has A6 To compare treatment to control you could compare A1 &A2 vs A4 & A6, A1 & A3 vs A5 & A6, A1 &A2 vs A5 &A6, A1&A3 vs A4&A6 Pro's: You are using all the data individually instead of averaging. Con's You can have lots of combinations (depending on the number of replicates) and the chances of getting a low p value in at least one of the combinations is increased compared to a gene that has no replicate probes, so you may be biasing the results (3) Randomly chose one of the technical replicates to represent a gene on a chip. ie randomly chose one of the combinations above for the analysis Pros: Not going to give some genes a higher chance of a low p values just by chance. Cons. Not using all the data I was wondering if any one had any thoughts on which of these alternatives is the best, or whether there is another alternative we haven't considered. Any ideas would be really appreciated! Thanks and Regards Marg Dr Margaret Gardiner-Garden Garvan Institute of Medical Research, Sydney Australia