Hello, I have cDNA, I tried limma to do normalizeWithinArrays, but the results looks like they did not aveage spots. Each spot was printed twice in my cDNA array, how limma average them? I have all the log ratio from every printed spot(twice printed). When I did correlation between replicated, their coef is very low, which compared to arrayTool analysis, is significant different. When I looked at the log ratio, limma has twice as many as arrayTool log ratio, so I think limma did not average my spots. Any idea and suggestion is greatly appreciated! Thanks ******************************** Joyce Gu M.S. Scientific Programmer Cancer Genomics Group Baylor College of Medicine Phone: 832-824-4680 Fax: 832-825-4038

Hi Joyce, The normalizeWithinArrays() function doesn't average across the duplicate spots (as its name suggests, it normalizes the log-ratios of all spots on each array). You can average over the duplicate spots by running the normalized log-ratios through lmFit() and specifying ndups=2, as well as a value for the 'spacing' argument (if the duplicate spots are side by side in the rows of the print-tip groups, spacing=1. If they are below each other, the spacing will be the number of spots in the row of a print-tip). You'll also need a design matrix, which will depend on your experiment, and what you're interested in (check out the limma user's guide for some examples). You might also want to use the duplicateCorrelation() function to estimate the spatial correlation between the duplicate spots. I've put an example of the code you might use below (if the duplicate spots were printed side-by-side). Best wishes, Matt Ritchie M <- normalizeWithinArrays(RG) cor <- duplicateCorrelation(M, design, ndups=2, spacing=1) # assuming you have specified a design matrix fit <- lmFit(M, design, ndups=2, spacing=1, correlation=cor$cor) aveM <- fit$coef > Hello, > I have cDNA, I tried limma to do normalizeWithinArrays, but the results > looks like they did not aveage spots. Each spot was printed twice in my > cDNA array, how limma average them? I have all the log ratio from every > printed spot(twice printed). When I did correlation between replicated, > their coef is very low, which compared to arrayTool analysis, is > significant different. When I looked at the log ratio, limma has twice > as many as arrayTool log ratio, so I think limma did not average my > spots. > > Any idea and suggestion is greatly appreciated! > > Thanks > > ******************************** > Joyce Gu M.S. > Scientific Programmer > > Cancer Genomics Group > Baylor College of Medicine > Phone: 832-824-4680 > Fax: 832-825-4038 > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor